Anti-cd100 antibodies and methods for using the same

ABSTRACT

Compositions and methods are provided for treating diseases associated with CD100, including certain autoimmune diseases, inflammatory diseases, and cancers. In particular, anti-CD100 monoclonal antibodies have been developed to neutralize CD100.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of currently pending U.S.application Ser. No. 13/800,713, filed Mar. 13, 2013, which is acontinuation of U.S. Non-Provisional application Ser. No. 12/776,187,filed on May 7, 2010, now U.S. Pat. No. 8,496,938, which claims thebenefit of U.S. Provisional Appl. No. 61/325,213, filed on Apr. 16,2010, and U.S. Provisional Appl. No. 61/176,826, filed on May 8, 2009.This application is also related to U.S. application Ser. No.13/517,807, filed on Jun. 14, 2012, now U.S. Pat. No. 8,816,058, and toU.S. Non-Provisional application Ser. No. 13/797,048, filed on Mar. 12,2012, now U.S. Pat. No.9,605,055. Each of these related applications ishereby incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name 165177_SequenceListing_ST25.txt; Size: 33,907 bytes; andDate of Creation: Apr. 28, 2017) filed with the application isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

CD100, also known as semaphorin 4D (SEMA4D), is a transmembrane protein(e.g., SEQ ID NO: 1 (human); SEQ ID NO: 2 (murine)) that belongs to thesemaphorin gene family. CD100 is expressed on the cell surface as ahomodimer, but upon cell activation CD100 can be released from the cellsurface via proteolytic cleavage to generate active sCD100, a solubleform of the protein. See Suzuki et al., Nature Rev. Immunol. 3:159-167(2003); Kukutani et al., Nature Immunol. 9:17-23 (2008).

CD100 was first identified by generating two mouse monoclonalantibodies, BD16 and BB18, against activated human T cell clones (Heroldet al., Int. Immunol. 7:1-8 (1994)). CD100 was the first example of asemaphorin expressed in the immune system. CD100 is expressed abundantlyon the surface of resting T cells, and weakly on resting B cells,monocytes, and professional antigen-presenting cells, such as dendriticcells (DCs). Cellular activation can stimulate up-regulation of surfaceexpression of CD100 on B cells and DCs, as well as the generation ofsCD100. CD100 is thought to function as both a receptor, which signalsthrough its cytoplasmic domain, and as a ligand (Hall et al., PNAS93:11780-11785 (1996)). One of the receptors identified for CD100 isPlexin-B1. Plexin-B1 is expressed in non-lymphoid tissues and is a highaffinity (1 nM) receptor for CD100 (Tamagnone et al., Cell 99:71-80(1999)).

CD100 is an important mediator of T cell and B cell activation. CD100knockout (CD100−/−) mice have reduced antibody responses to T-dependentantigens and impaired T cell priming. Both of these functions arerestored upon the administration of sCD100 (Shi et al., Immunity13:633-642 (2000)).

In addition to the demonstrated effects of CD100 on immune cells, CD100also appears to play a direct role in the demyelination and axonaldegeneration seen in neuroinflammatory diseases. The pathogenesis ofinflammatory demyelinating diseases, such as MS, includes both aninflammatory phase involving immune cells as well as phases of selectivedemyelination and neurodegeneration. CD100 is expressed in centralnervous system (CNS) oligodendrocytes and is an inhibitor of axonalregeneration. CD100 expression is up-regulated in oligodendrocytes atthe periphery of spinal cord lesions (Moreau-Fauvarque et al., J.Neuroscience 23:9229-9239 (2003)). Culturing chronically activated Tcells expressing sCD100 with human multipotent neural precursors orprimary oligodendrocytes from rat brain induces apoptosis and processextension collapse (Giraudon et al., J. Immunol. 172:1246-1255 (2004);Giraudon et al., NeuroMolecular Med. 7:207-216 (2005)). CD100 inducedapoptosis of neural precursors can be inhibited by the BD16 anti-CD100antibody.

CD100 knockout mice are resistant to the development of experimentalallergic encephalomyelitis (EAE), which is a mouse model for humanmultiple sclerosis (MS) (Kumanogoh et al., J. Immunol. 169:1175-1181(2002)).

A number of other studies have demonstrated that CD100 induces growthcone collapse in neurons, and, in further support of the functionalrelevance of CD100 in neuroinflammation, it has been reported that thereare highly elevated levels of sCD100 in cerebrospinal fluid (CSF) ofHTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP)patients. Thus, there is a direct deleterious effect of sCD100 onoligodendrocyte and neural precursor integrity and CD100 may play apathogenic role in demyelination. As an important mediator of bothinflammatory responses and direct demyelination, there is a need in theart for CD100 neutralizing molecules, e.g., anti-CD100 antibodies, fortreatment of inflammatory and demyelinating diseases.

CD100 is also a potent pro-angiogenic molecule. Activation of Plexin-B1through CD100 binding transactivates c-Met and promotes the invasiveability of tumor cells and promotes angiogenesis both in vitro and invivo. Immunohistochemical analysis of CD100 in a large tumor samplecollection revealed that CD100 overexpression is a very frequent eventin head and neck, prostate, colon, breast, and lung cancers.

CD100/Plexin B1 signaling has also been shown to induce migration ofendothelial cells and to promote migration of tumor cells (Conrotto etal., Blood 105:4321-4329 (2005); Giordano et al., Nature Cell Biology4:720-724 (2002)). CD100 induced endothelial cell migration is preventedby CD100-blocking antibodies and by CD100 knockdown. Knocking down CD100expression in head and neck squamous cell carcinoma (HNSCC) cells withCD100 short hairpin RNA (shRNA) before grafting into nude mice caused adramatic reduction in tumor vascularity and tumor growth (Basile et al.,PNAS 103:9017-9022 (2006)). Reports have recently pointed to a closecorrelation between inflammatory infiltration of the tumor stroma and ahigh vascular grade. CD100 is produced by inflammatory cells present inthe tumor microenvironment. In an environment lacking CD100, the abilityof mouse breast cancer cells to originate tumor masses and metastaseswas severely impaired, and the source of CD100 was tumor associatedmacrophages (Sierra et al., JEM 205:1673-1685 (2008)). Thus, there is afurther need in the art for CD100 neutralizing molecules, e.g.,anti-CD100 antibodies, for the treatment of CD100 cancer.

FIELD OF THE INVENTION

The invention relates to CD100 neutralizing antibodies, e.g., humanizedmonoclonal antibodies, methods of using the antibodies, and methods fortreatment of conditions and diseases associated with CD100-expressingcells.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods are provided for treating diseases associatedwith CD100, including certain such as certain types of autoimmunediseases, inflammatory diseases, cancers and invasive angiogenesis. Inparticular, anti-CD100 monoclonal antibodies have been developed toneutralize CD100. Mouse MAb 67 demonstrated the ability to block CD100activity in vitro, and, reduce the severity of clinical signs ofexperimental allergic encephalomyelitis (EAE), collagen-inducedarthritis (CIA), and cancer in mouse models. MAb 2503 is a humanizedversion of MAb 67 which has demonstrated improved affinity to human andmurine CD100 and similar CD100 blocking activity as MAb 67.

In one embodiment, the invention provides an isolated binding moleculewhich specifically binds to the same CD100 epitope as a referencemonoclonal antibody selected from the group consisting of 2503, 67, or76.

In another embodiment, the invention provides an isolated bindingmolecule which specifically binds to CD100, wherein said bindingmolecule competitively inhibits a reference monoclonal antibody selectedfrom the group consisting of 2503, 67, or 76 from specifically bindingto CD100.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof which specifically binds to CD100,wherein said antibody or fragment thereof is monoclonal antibody 2503,67, or 76.

In certain embodiments, the isolated antibody or antigen-bindingfragment thereof of the invention which specifically binds to CD100,comprises a heavy chain variable region (VH) that has an amino acidsequence at least 90% identical to SEQ ID NO: 9, SEQ ID NO: 10, or SEQID NO: 25. In another aspect of the invention, the VH of said antibodyor fragment thereof comprises an amino acid sequence identical, exceptfor 20 or fewer conservative amino acid substitutions, to SEQ ID NO: 9,SEQ ID NO: 10, or SEQ ID NO: 25. In yet another aspect of the invention,the VH of said antibody or fragment thereof comprises or consists of theamino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25.

In certain embodiments, the isolated antibody or antigen-bindingfragment thereof of the invention which specifically binds to CD100,comprises a light chain variable region (VL) that has an amino acidsequence at least 90% identical to SEQ ID NO: 17, SEQ ID NO: 18, or SEQID NO: 29. In another aspect of the invention, the VL of said antibodyor fragment thereof comprises an amino acid sequence identical, exceptfor 20 or fewer conservative amino acid substitutions, to SEQ ID NO: 17,SEQ ID NO: 18, or SEQ ID NO: 29. In yet another aspect of the invention,the VL of said antibody or fragment thereof comprises or consists of theamino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 29.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof which specifically binds to CD100,wherein the VH of said antibody or fragment thereof comprises at leastone of the following CDRs: a Chothia-Kabat heavy chain complementaritydetermining region-1 (VH-CDR1) amino acid sequence identical, except fortwo or fewer amino acid substitutions, to SEQ ID NO: 6, a Kabat heavychain complementarity determining region-2 (VH-CDR2) amino acid sequenceidentical, except for four or fewer amino acid substitutions, to SEQ IDNO: 7, or a Kabat heavy chain complementarity determining region-3(VH-CDR3) amino acid sequence identical, except for two or fewer aminoacid substitutions, to SEQ ID NO: 8.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof which specifically binds to CD100,wherein the VL of said antibody or fragment thereof comprises at leastone of the following CDRs: a Kabat light chain complementaritydetermining region-1 (VL-CDR1) amino acid sequence identical, except forfour or fewer amino acid substitutions, to SEQ ID NO: 14, a Kabat lightchain complementarity determining region-2 (VL-CDR2) amino acid sequenceidentical, except for two or fewer amino acid substitutions, to SEQ IDNO: 15, or a Kabat light chain complementarity determining region-3(VL-CDR3) amino acid sequence identical, except for two or fewer aminoacid substitutions, to SEQ ID NO: 16.

In another aspect, the VH of an antibody or fragment thereof of theinvention comprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequencescomprising SEQ ID NOs: 6, 7, and 8, respectively, except for four orfewer amino acid substitutions in one or more of said VH-CDRs. In afurther aspect, the VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequencesare SEQ ID NOs: 6, 7, and 8, respectively.

In another aspect, the VL of an antibody or fragment thereof of theinvention comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequencescomprising SEQ ID NOs: 14, 15, and 16, respectively, except for four orfewer amino acid substitutions in one or more of said VL-CDRs. In afurther aspect, the VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequencesare SEQ ID NOs: 14, 15, and 16, respectively.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof which specifically binds to CD100,wherein the VH of said antibody or fragment thereof comprises at leastone of the following CDRs: a Kabat heavy chain complementaritydetermining region-1 (VH-CDR1) amino acid sequence identical, except fortwo or fewer amino acid substitutions, to SEQ ID NO: 26, a Kabat heavychain complementarity determining region-2 (VH-CDR2) amino acid sequenceidentical, except for four or fewer amino acid substitutions, to SEQ IDNO: 27, or a Kabat heavy chain complementarity determining region-3(VH-CDR3) amino acid sequence identical, except for two or fewer aminoacid substitutions, to SEQ ID NO: 28.

In another embodiment, the invention provides an isolated antibody orantigen-binding fragment thereof which specifically binds to CD100,wherein the VL of said antibody or fragment thereof comprises at leastone of the following CDRs: a Kabat light chain complementaritydetermining region-1 (VL-CDR1) amino acid sequence identical, except forfour or fewer amino acid substitutions, to SEQ ID NO: 30, a Kabat lightchain complementarity determining region-2 (VL-CDR2) amino acid sequenceidentical, except for two or fewer amino acid substitutions, to SEQ IDNO: 31, or a Kabat light chain complementarity determining region-3(VL-CDR3) amino acid sequence identical, except for two or fewer aminoacid substitutions, to SEQ ID NO: 32.

In another aspect, the VH of an antibody or fragment thereof of theinvention comprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequencescomprising SEQ ID NOs: 26, 27, and 28, respectively, except for four orfewer amino acid substitutions in one or more of said VH-CDRs. In afurther aspect, the VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequencesare SEQ ID NOs: 26, 27, and 28, respectively.

In another aspect, the VL of an antibody or fragment thereof of theinvention comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequencescomprising SEQ ID NOs: 30, 31, and 32, respectively, except for four orfewer amino acid substitutions in one or more of said VL-CDRs. In afurther aspect, the VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequencesare SEQ ID NOs: 30, 31, and 32, respectively.

In another aspect, an antibody or fragment thereof of the inventionbinds to human and murine CD100. In another aspect, the antibody orfragment thereof of the invention specifically binds to an CD100polypeptide or fragment thereof, or a CD100 variant polypeptide with anaffinity characterized by a dissociation constant (K_(D)) no greaterthan 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M,10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 5.7×10⁻¹² M,8.4×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M,or 10⁻¹⁵ M. In certain aspects, the CD100 polypeptide or fragmentthereof, or a CD100 variant polypeptide is human or murine. In furtheraspects, a CD100 polypeptide or fragment thereof, or a CD100 variantpolypeptide is human and said K_(D) is about 5×10⁻⁹ M to about 6×10⁻⁹ M.In yet another aspect, a CD100 polypeptide or fragment thereof, or aCD100 variant polypeptide is murine and said K_(D) is about 1×10⁻⁹ M toabout 2×10⁻⁹ M.

In another aspect, the antibody or fragment thereof of the invention ishumanized, primatized or chimeric.

In another embodiment, the invention provides a composition comprisingan antibody or fragment thereof of the invention, and a carrier.

In another embodiment, the invention provides an isolated polynucleotidecomprising a nucleic acid which encodes an antibody VH or VL polypeptideof the invention. In another aspect, the polynucleotide of the inventioncomprises or consists of a nucleic acid which encodes an antibody orfragment thereof of the invention. In yet another aspect, the inventionprovides a vector comprising a polynucleotide of the invention. Inanother aspect, the invention provides a host cell comprising the vectorof the invention. In another aspect, the invention provides a method ofproducing an antibody of the invention.

In another embodiment, the invention provides a method for treating anautoimmune disease or an inflammatory disease in an animal in need oftreatment, comprising administering to said animal a compositioncomprising: the isolated antibody or fragment thereof of the inventionand a pharmaceutically acceptable carrier. In further embodiments, theautoimmune disease or inflammatory disease is multiple sclerosis orarthritis.

In another embodiment, the invention provides a method for treating acancer in an animal in need of treatment, comprising administering tosaid animal a composition comprising: the isolated antibody or fragmentthereof of the invention and a pharmaceutically acceptable carrier.

In another embodiment, the invention provides a method for inhibitingangiogenesis in an animal in need of treatment for cancer, comprisingadministering to said animal a composition comprising: the isolatedantibody or fragment thereof of the invention and a pharmaceuticallyacceptable carrier.

In a further aspect, the antibody or fragment thereof of the inventioninhibits CD100 binding to a CD100 receptor. In yet another aspect of theinvention, the CD100 receptor is Plexin-B 1.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Diagram of CD100 blocking assay. CD100-His shown binding toPlexin B1 on the cell surface of a stable cell line expressing Plexin B1(293/Plexin). The CD100-His which is bound to Plexin B1 is detectedusing a biotin conjugated anti-His tag specific monoclonal antibody andstreptavidin-APC. Anti-CD100 MAbs which are able to block binding ofCD100-His to Plexin B1 result in lower fluorescence associated with the293/Plexin cells as measured by flow cytometry.

FIG. 2. Flow cytometry results for rabbit anti-His+streptavidin-APC (Rbanti-his+sAPC), mouse CD100 (muCD100 only), mouse CD100+0.625 μg/ml MAb(MAb 67, MAb 76, and mIgG isotype), and mouse CD100+0.156 μg/ml MAb (MAb67, MAb 76, and mIgG isotype) tested in the CD100 blocking assaydescribed in FIG. 1 are shown. Monoclonal antibodies 67 and 76 blockmouse CD100 binding to Plexin B1 receptor.

FIG. 3. Monoclonal antibodies 67 and 76 block mouse CD100 mediateddetachment of 293/Plexin B cells from a fibronectin coated plate, asshown by an increase in absorbance for both MAbs 67 (67-2) and 76 (76-1)compared to isotype control.

FIG. 4A and FIG. 4B. Treatment with 30 mg/kg anti-CD100 MAb 76 (1×/weekor 2×/week) or MAb 67 (1×/week or 2×/week) attenuates relapsingremitting EAE in SJL mice compared to treatment with mouse IgG controlas shown by reduction in clinical score (FIG. 4A). The results arefurther illustrated by comparing percent reduction in Group Mean Score(GMS) for each MAb treatment between day 21 and study end (FIG. 4B).

FIG. 5A and FIG. 5B. Treatment with 30 mg/kg anti-CD100 MAb 76 (1×/week)or MAb 67 (1×/week) attenuates relapsing remitting EAE in SJL micecompared to treatment with mouse IgG control as shown by reduction inclinical score (FIG. 5A). The results are further illustrated bycomparing percent reduction in Group Mean Score (GMS) for both MAbtreatments between day 18 and study end (FIG. 5B).

FIG. 6. Treatment with 30 mg/kg anti-CD100 MAb 67 starting at day 7post-immunization (1×/week) attenuates relapsing remitting EAE in SJLmice compared to treatment with mouse IgG control as shown by reductionin clinical score.

FIG. 7A and FIG. 7B. ELISA results showing percent (%) blocking ofbiotinylated 67 binding to human CD100 (FIG. 7A) or mouse CD100 (FIG.7B) due to competitive binding of MAb 2503, MAb 67, or IgG control.

FIG. 8A, FIG. 8B, and FIG. 8C. Flow cytometry results forstreptavidin-APC (sAPC only), human CD100 (huCD100), marmoset CD100(marmCD100), mouse CD100 (muCD100), 1.0 μg isotype, and 1.0 μg MAb (67or 2503) tested in the CD100 blocking assay described in FIG. 1 areshown. MAb 67 and MAb 2503 block human CD100 (FIG. 8A), marmoset (FIG.8B), or mouse (FIG. 8C) CD100 from binding to Plexin B1 receptor.

FIG. 9A and FIG. 9B. A blocked reduction in absorbance caused by CD100due to neutralization of CD100 by MAb 67, MAb 2503, and IgG control isshown. Anti-CD100 MAb 67 and MAb 2503 block human CD100 (FIG. 9A) andmarmoset CD100 (FIG. 9B) mediated detachment of 293/Plexin cells from afibronectin coated plate.

FIG. 10. Change in tumor volume (mm³) is shown for wild-type Balb/c miceand CD100−/− mice after 50,000 CT26 colon tumor cells were injected intothe leg muscle of the mice.

FIG. 11. Change in mean leg volume (mm³) is shown for wild-type Balb/cmice treated with lmg MAb 67 or lmg control mouse IgG and CD100−/− mice(“KO”) after 50,000 CT26 tumor cells were injected into the leg muscleof the mice.

FIG. 12. A schematic showing a general treatment strategy for CollagenInduced Arthritis (CIA)

FIG. 13A and FIG. 13B. Reduction in arthritis disease development in CIAmodel was shown for groups treated with 600 μg MAb 67. Arthritic Index(AI) in mice treated with 600 μg MAb 67 was compared to AI in micetreated with 600 μg negative control (IgG1) and 600 μg positive controletanercept (Enbrel®) when treatment was started at day 20 (FIG. 13A).Arthritic Index (AI) results for treatment with MAb 67 were compared totreatment with a negative control (IgG1) and positive control etanercept(EMBREL®) when treatment was started either at day 20 or when the AI was≧3 (FIG. 13B).

FIG. 14A and FIG. 14B. In Balb/c mice immunized with(4-hydroxy-3-nitrophenyl) acetyl conjugated chicken gamma globulinprecipitated with alum (aluminum-/magnesium-hydroxide) (“NP-CGG”),treatment with 600 μg MAb 67 decreased the number of germinal center(GC) B cells (“B220+CD38lowPNA+”) in spleen (SP) and lymph nodes (LN)after both primary immunization (FIG. 14A) and secondary immunization(FIG. 14B). Results are also shown for CD100 −/− mice and Balb/c micewith and without NP-CGG immunization.

FIG. 15. Change in tumor volume (mm³) is shown for wild-type Balb/c miceafter 50,000 CT26 colon tumor cells were injected into the leg muscle ofthe mice. Results are shown for mice injected with 1 mg MAb 67 weeklystarting on day 1 compared to mice injected with IgG control. The studywas carried out to an end point of tumor growth delay.

FIG. 16A and FIG. 16B. Change in tumor volume (mm³) is shown forwild-type Balb/c mice and CD100−/− mice (“SEMA4D−/−”) after 50,000 BCA34fibroblastic tumor cells were s.c. injected into the abdominal region ofthe mice (FIG. 16A). Change in mean thigh volume (mm³) is shown forwild-type Balb/c mice treated with 1 mg MAb 67 or 1 mg control mouse IgGafter 50,000 BCA34 fibroblastic tumor cells were injected into the legmuscle of the mice (FIG. 16B).

FIG. 17. Change in tumor volume (mm³) is shown for wild-type Balb/c miceand CD100−/− mice (“SEMA4D−/−”) after 50,000 EMT6 mouse mammarycarcinoma tumor cells were injected into the leg muscle of the mice.

FIG. 18. Change in tumor volume (mm³) is shown for athymic nude miceafter two HN12 head and neck tumors/mouse were s.c. injected into theflank muscle of the mice. Results are shown for mice injected with 1 mgMAb 2503 weekly starting on day 1 post graft compared to mice injectedwith IgG4 control.

FIG. 19A and FIG. 19B. Change in tumor volume (mm³) is shown for athymicnude mice after two HN6 HIF1a mODD head and neck tumors were s.c.injected into the leg muscle of the mice. Results are shown for miceinjected with 1 mg MAb 2503 weekly starting on day 1 post graft comparedto mice injected with IgG4 control (FIG. 19A). Pictures ofrepresentative tumors from IgG4 control and MAb 2503 treated mice areshown (FIG. 19B).

FIG. 20A and FIG. 20B. Percent saturation results from singleintravenous injection saturation analysis of MAb 2503 in rat.Sprague-Dawley rats were administered a single intravenous injection ofMAb 2503 at doses of 0, 0.01, 0.1, 1.0, 10, and 100 mg/kg. A flowcytometry-based saturation assay was performed on lysed whole blood atvarious time points to determine the percent of the cellular target(SEMA4D) that was saturated with MAb 2503 in male (FIG. 20A) and female(FIG. 20B) rats.

FIG. 21. Percent saturation results from single intravenous injectionsaturation analysis of MAb 2503 in cynomolgus monkey. Cynomolgus monkeyswere administered a single intravenous injection of MAb 2503 at doses of0, 0.01, 0.1, 1.0, 10, and 100 mg/kg. A flow cytometry-based saturationassay was performed on lysed whole blood at various time points todetermine the percent of the cellular target (SEMA4D) that was saturatedwith MAb 2503 (male and female data were combined).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an anti-CD100 antibody” is understood torepresent one or more anti-CD100 antibodies. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all cancerous andpre-cancerous cells and tissues.

“Invasive angiogenesis” refers to the formation of blood vessels for thesupport of pathological conditions, including malignant andnon-malignant tumors as well as the abnormal formation of new bloodvessels in macular degeneration.

The terms, “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto carcinomas, lymphomas and leukemias.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides that do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purpose of the invention, as are native orrecombinant polypeptides that have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative,” and“analog” when referring to anti-CD100 antibodies or antibodypolypeptides of the present invention include any polypeptides thatretain at least some of the antigen-binding properties of thecorresponding antibody or antibody polypeptide of the invention.Fragments of polypeptides of the present invention include proteolyticfragments, as well as deletion fragments, in addition to specificantibody fragments discussed elsewhere herein. Variants of anti-CD100antibodies and antibody polypeptides of the present invention includefragments as described above, and also polypeptides with altered aminoacid sequences due to amino acid substitutions, deletions, orinsertions. Variants may occur naturally or be non-naturally occurring.Non-naturally occurring variants may be produced using art-knownmutagenesis techniques. Variant polypeptides may comprise conservativeor non-conservative amino acid substitutions, deletions, or additions.Variant polypeptides may also be referred to herein as “polypeptideanalogs.” As used herein a “derivative” of an anti-CD100 antibody orantibody polypeptide refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Also included as “derivatives” are those peptides that contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For example, 4-hydroxyproline may be substituted forproline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Derivatives of anti-CD100 antibodies and antibody polypeptides of thepresent invention, may include polypeptides that have been altered so asto exhibit additional features not found on the reference antibody orantibody polypeptide of the invention.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, that has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan anti-CD100 binding molecule, e.g., an antibody or antigen bindingfragment thereof, contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, a polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid thatconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding ananti-CD100 antibody or fragment, variant, or derivative thereof.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid that encodesa polypeptide normally may include a promoter and/or other transcriptionor translation control elements operably associated with one or morecoding regions. An operable association is when a coding region for agene product, e.g., a polypeptide, is associated with one or moreregulatory sequences in such a way as to place expression of the geneproduct under the influence or control of the regulatory sequence(s).Two DNA fragments (such as a polypeptide coding region and a promoterassociated therewith) are “operably associated” if induction of promoterfunction results in the transcription of mRNA encoding the desired geneproduct and if the nature of the linkage between the two DNA fragmentsdoes not interfere with the ability of the expression regulatorysequences to direct the expression of the gene product or interfere withthe ability of the DNA template to be transcribed. Thus, a promoterregion would be operably associated with a nucleic acid encoding apolypeptide if the promoter was capable of effecting transcription ofthat nucleic acid. The promoter may be a cell-specific promoter thatdirects substantial transcription of the DNA only in predeterminedcells. Other transcription control elements, besides a promoter, forexample enhancers, operators, repressors, and transcription terminationsignals, can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions that function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited to,ribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions that encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence that is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

A “binding molecule” or “antigen binding molecule” of the presentinvention refers in its broadest sense to a molecule that specificallybinds an antigenic determinant. In one embodiment, the binding moleculespecifically binds to CD100, e.g., a transmembrane CD100 polypeptide ofabout 150 kDa or a soluble CD100 polypeptide of about 120 kDa (commonlyreferred to as sCD100). In another embodiment, a binding molecule of theinvention is an antibody or an antigen binding fragment thereof. Inanother embodiment, a binding molecule of the invention comprises atleast one heavy or light chain CDR of an antibody molecule. In anotherembodiment, a binding molecule of the invention comprises at least twoCDRs from one or more antibody molecules. In another embodiment, abinding molecule of the invention comprises at least three CDRs from oneor more antibody molecules. In another embodiment, a binding molecule ofthe invention comprises at least four CDRs from one or more antibodymolecules. In another embodiment, a binding molecule of the inventioncomprises at least five CDRs from one or more antibody molecules. Inanother embodiment, a binding molecule of the invention comprises atleast six CDRs from one or more antibody molecules.

The present invention is directed to certain anti-CD100 antibodies, orantigen-binding fragments, variants, or derivatives thereof. Unlessspecifically referring to full-sized antibodies such as naturallyoccurring antibodies, the term “anti-CD100 antibodies” encompassesfull-sized antibodies as well as antigen-binding fragments, variants,analogs, or derivatives of such antibodies, e.g., naturally occurringantibody or immunoglobulin molecules or engineered antibody molecules orfragments that bind antigen in a manner similar to antibody molecules.

As used herein, “human” or “fully human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example, in U.S.Pat. No. 5,939,598 by Kucherlapati et al. “Human” or “fully human”antibodies also include antibodies comprising at least the variabledomain of a heavy chain, or at least the variable domains of a heavychain and a light chain, where the variable domain(s) have the aminoacid sequence of human immunoglobulin variable domain(s).

“Human” or “fully human” antibodies also include “human” or “fullyhuman” antibodies, as described above, that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or fragments thereof immunospecifically bind toa CD100 polypeptide or fragment or variant thereof. Standard techniquesknown to those of skill in the art can be used to introduce mutations inthe nucleotide sequence encoding a human anti-CD100 antibody, including,but not limited to, site-directed mutagenesis and PCR-mediatedmutagenesis which result in amino acid substitutions. Preferably, thevariants (including derivatives) encode less than 50 amino acidsubstitutions, less than 40 amino acid substitutions, less than 30 aminoacid substitutions, less than 25 amino acid substitutions, less than 20amino acid substitutions, less than 15 amino acid substitutions, lessthan 10 amino acid substitutions, less than 5 amino acid substitutions,less than 4 amino acid substitutions, less than 3 amino acidsubstitutions, or less than 2 amino acid substitutions relative to thereference VH region, VHCDR1, VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2,or VLCDR3.

In certain embodiments, the amino acid substitutions are conservativeamino acid substitution, discussed further below. Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind a CD100 polypeptide, e.g., human,murine, or both human and murine CD100). Such variants (or derivativesthereof) of “human” or “fully human” antibodies can also be referred toas human or fully human antibodies that are “optimized” or “optimizedfor antigen binding” and include antibodies that have improved affinityto antigen.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory Press).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL or VK) and heavy (VH) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs) within these variable domains, of an antibodycombine to form the variable region that defines a three dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains. In some instances, e.g., certain immunoglobulinmolecules derived from camelid species or engineered based on camelidimmunoglobulins, a complete immunoglobulin molecule may consist of heavychains only, with no light chains. See, e.g., Hamers-Casterman et al.,Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a (β-sheet conformation and the CDRsform loops that connect, and in some cases form part of, the (β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable domainby one of ordinary skill in the art, since they have been preciselydefined (see below).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al. (1983) U.S. Dept. of Health and HumanServices, “Sequences of Proteins of Immunological Interest” and byChothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which areincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residuesthat encompass the CDRs as defined by each of the above cited referencesare set forth below in Table 1 as a comparison. The exact residuenumbers that encompass a particular CDR will vary depending on thesequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al. (1983) U.S. Dept. ofHealth and Human Services, “Sequence of Proteins of ImmunologicalInterest.” Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in an anti-CD100 antibody orantigen-binding fragment, variant, or derivative thereof of the presentinvention are according to the Kabat numbering system.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single-chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv),disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto anti-CD100 antibodies disclosed herein). ScFv molecules are known inthe art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulin molecule.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain anti-CD100 antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein, the heavy chainportions of one polypeptide chain of a multimer are identical to thoseon a second polypeptide chain of the multimer. Alternatively, heavychain portion-containing monomers of the invention are not identical.For example, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding molecule for use in the diagnosticand treatment methods disclosed herein may be derived from differentimmunoglobulin molecules. For example, a heavy chain portion of apolypeptide may comprise a C_(H1) domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain, e.g., a kappa orlambda light chain. Preferably, the light chain portion comprises atleast one of a VL or CL domain.

Anti-CD100 antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein may be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide disclosed herein (e.g., CD100) that they recognize orspecifically bind. The portion of a target polypeptide that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target polypeptide maycomprise a single epitope, but typically comprises at least twoepitopes, and can include any number of epitopes, depending on the size,conformation, and type of antigen. Furthermore, it should be noted thatan “epitope” on a target polypeptide may be or may includenon-polypeptide elements, e.g., an epitope may include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. A peptide or polypeptide epitope recognized by anti-CD100antibodies of the present invention may contain a sequence of at least4, at least 5, at least 6, at least 7, more preferably at least 8, atleast 9, at least 10, at least 15, at least 20, at least 25, or betweenabout 15 to about 30 contiguous or non-contiguous amino acids of CD100.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

By way of non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody may beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

In another non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody may be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody may be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope. An antibody or antigen-binding fragment, variant, orderivative disclosed herein may be said to bind a target polypeptidedisclosed herein (e.g., CD100, e.g., human, murine, or both human andmurine CD100) or a fragment or variant thereof with an off rate (k(off))of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³sec⁻¹. More preferably, an antibody of the invention may be said to binda target polypeptide disclosed herein (e.g., CD100, e.g., human, murine,or both human and murine CD100) or a fragment or variant thereof with anoff rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷sec⁻¹.

An antibody or antigen-binding fragment, variant, or derivativedisclosed herein may be said to bind a target polypeptide disclosedherein (e.g., CD100, e.g., human, murine, or both human and murineCD100) or a fragment or variant thereof with an on rate (k(on)) ofgreater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of the invention may besaid to bind a target polypeptide disclosed herein (e.g., CD100, e.g.,human, murine, or both human and murine CD100) or a fragment or variantthereof with an on rate (k(on)) greater than or equal to 10⁵ M⁻¹ sec⁻¹,5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

Anti-CD100 antibodies or antigen-binding fragments, variants, orderivatives thereof of the invention may also be described or specifiedin terms of their cross-reactivity. As used herein, the term“cross-reactivity” refers to the ability of an antibody, specific forone antigen, to react with a second antigen; a measure of relatednessbetween two different antigenic substances. Thus, an antibody is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, may actually fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Anti-CD100 binding molecules, e.g., antibodies or antigen-bindingfragments, variants or derivatives thereof, of the invention may also bedescribed or specified in terms of their binding affinity to apolypeptide of the invention, e.g., CD100, e.g., human, murine, or bothhuman and murine CD100. Preferred binding affinities include those witha dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M. In certain embodiments, the anti-CD100 bindingmolecule, e.g., an antibody or antigen binding fragment thereof, of theinvention binds human CD100 with a Kd of about 5×10⁻⁹ to about 6×10⁻⁹.In another embodiment, the anti-CD100 binding molecule, e.g., anantibody or antigen binding fragment thereof, of the invention bindsmurine CD100 with a Kd of about 1×10⁻⁹ to about 2×10⁻⁹.

Anti-CD100 antibodies or antigen-binding fragments, variants orderivatives thereof of the invention may be “multispecific,” e.g.,bispecific, trispecific, or of greater multispecificity, meaning that itrecognizes and binds to two or more different epitopes present on one ormore different antigens (e.g., proteins) at the same time. Thus, whetheran anti-CD100 antibody is “monospecific” or “multispecific,” e.g.,“bispecific,” refers to the number of different epitopes with which abinding polypeptide reacts. Multispecific antibodies may be specific fordifferent epitopes of a target polypeptide described herein or may bespecific for a target polypeptide as well as for a heterologous epitope,such as a heterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains present in a bindingpolypeptide or CD100 binding molecule, e.g., an antibody or antigenbinding fragment thereof. Each binding domain specifically binds oneepitope. When a binding polypeptide or CD100 binding molecule comprisesmore than one binding domain, each binding domain may specifically bindthe same epitope, for an antibody with two binding domains, termed“bivalent monospecific,” or to different epitopes, for an antibody withtwo binding domains, termed “bivalent bispecific.” An antibody orantigen binding fragment thereof may also be bispecific and bivalent foreach specificity (termed “bispecific tetravalent antibodies”). Inanother embodiment, tetravalent minibodies or domain deleted antibodiescan be made.

Bispecific bivalent antibodies, and methods of making them, aredescribed, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Patent Appl. Publ. Nos. 2003/020734 and2002/0155537, the disclosures of all of which are incorporated byreference herein. Bispecific tetravalent antibodies, and methods ofmaking them are described, for instance, in WO 02/096948 and WO00/44788, the disclosures of both of which are incorporated by referenceherein. See generally, PCT publications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S.Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;Kostelny et al., J. Immunol. 148: 1547-1553 (1992).

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat EA et al.). The CH2 domain is unique in that it is not closelypaired with another domain. Rather, two N-linked branched carbohydratechains are interposed between the two CH2 domains of an intact nativeIgG molecule. It is also well documented that the CH3 domain extendsfrom the CH2 domain to the C-terminal of the IgG molecule and comprisesapproximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J.Immunol. 161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In preferred embodiments the targetbinding region or site will be from a non-human source (e.g., mouse orprimate) and the constant region is human (for example, monoclonalantibody (MAb) 2368 described herein).

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy or light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable domain to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site.

It is further recognized that the framework regions within the variabledomain in a heavy or light chain, or both, of a humanized antibody maycomprise solely residues of human origin, in which case these frameworkregions of the humanized antibody are referred to as “fully humanframework regions” (for example, MAb 2503). Alternatively, one or moreresidues of the framework region(s) of the donor variable domain can beengineered within the corresponding position of the human frameworkregion(s) of a variable domain in a heavy or light chain, or both, of ahumanized antibody if necessary to maintain proper binding or to enhancebinding to the CD100 antigen. A human framework region that has beenengineered in this manner would thus comprise a mixture of human anddonor framework residues, and is referred to herein as a “partiallyhuman framework region.”

For example, humanization of an anti-CD100 antibody can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodentor mutant rodent CDRs or CDR sequences for the corresponding sequencesof a human anti-CD100 antibody. See also U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; 5,693,762; 5,859,205; herein incorporated byreference. The resulting humanized anti-CD100 antibody would comprise atleast one rodent or mutant rodent CDR within the fully human frameworkregions of the variable domain of the heavy and/or light chain of thehumanized antibody. In some instances, residues within the frameworkregions of one or more variable domains of the humanized anti-CD100antibody are replaced by corresponding non-human (for example, rodent)residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762; and 6,180,370), in which case the resulting humanizedanti-CD100 antibody would comprise partially human framework regionswithin the variable domain of the heavy and/or light chain.

Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature 331:522-525(1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992); herein incorporated by reference.Accordingly, such “humanized” antibodies may include antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some framework residues are substitutedby residues from analogous sites in rodent antibodies. See, for example,U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205.See also U.S. Pat. No. 6,180,370, and International Publication No. WO01/27160, where humanized antibodies and techniques for producinghumanized antibodies having improved affinity for a predeterminedantigen are disclosed.

As used herein, the terms “linked,” “fused,” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of multiplesclerosis, arthritis, or cancer. Beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-CD100 antibody” and “an animal in need oftreatment” includes subjects, such as mammalian subjects, that wouldbenefit from administration of an anti-CD100 antibody used, e.g., fordetection of an anti-CD100 polypeptide (e.g., for a diagnosticprocedure) and/or from treatment, i.e., palliation or prevention of adisease, with an anti-CD100 antibody. As described in more detailherein, an anti-CD100 antibody can be used in unconjugated form or canbe conjugated, e.g., to a drug, prodrug, or an isotope.

II. Target Polypeptide Description

As used herein, the terms “CD100” and “CD100 polypeptide” are usedinterchangeably. In certain embodiments, CD100 is expressed on thesurface of or secreted by a cell. In another embodiment, CD100 ismembrane bound. In another embodiments, CD100 is soluble, e.g., sCD100.In another embodiments, CD100 may include a full-sized CD100 or afragment thereof, or a CD100 variant polypeptide, wherein the fragmentof CD100 or CD100 variant polypeptide retains some or all functionalproperties of the full-sized CD100.

The full-sized human CD100 protein is a homodimeric transmembraneprotein consisting of two polypeptide chains of 150 kDa. CD100 belongsto the semaphorin family of cell surface receptors and is also referredto as SEMA4D. Both human and mouse Sema4D/CD100 are proteolyticallycleaved from their transmembrane form to generate 120-kDa soluble forms,indicating the existence of two Sema4D isoforms (Kumanogoh et al., J.Cell Science 116(7):3464 (2003)). Semaphorins consist of soluble andmembrane-bound proteins that were originally defined as axonal-guidancefactors which play an important role in establishing precise connectionsbetween neurons and their appropriate target. Structurally considered aclass IV semaphorin, CD100 consists of an amino-terminal signal sequencefollowed by a characteristic ‘Sema’ domain, which contains 17 conservedcysteine residues, an Ig-like domain, a lysine-rich stretch, ahydrophobic transmembrane region, and a cytoplasmic tail.

Each polypeptide chain of CD100 includes a signal sequence of about 13amino acids followed by a semaphorin domain of about 512 amino acids, animmunoglobulin-like (Ig-like) domain of about 65 amino acids, alysine-rich stretch of 104 amino acids, a hydrophobic transmembraneregion of about 19 amino acids, and a cytoplasmic tail of 110 aminoacids. A consensus site for tyrosine phosphorylation in the cytoplasmictail supports the predicted association of CD100 with a tyrosine kinase(Schlossman, et al., Eds. (1995) Leucocyte Typing V (Oxford UniversityPress, Oxford).

Two types of receptors have been identified for CD100. One of thereceptors, Plexin-B1, is expressed in non-lymphoid tissues and has beenshown to be a high affinity (1 nM) receptor for CD100 (Tamagnone et al.,Cell 99:71-80 (1999)). CD100 stimulation of Plexin B1 signaling has beenshown to induce growth cone collapse of neurons, and to induce processextension collapse and apoptosis of oligodendrocytes (Giraudon et al.,J. Immunol. 172:1246-1255 (2004); Giraudon et al., NeuroMolecular Med.7:207-216 (2005)). After binding to CD100, Plexin B1 signaling mediatesthe inactivation of R-Ras, leading to a decrease in the integrinmediated attachment to the extracellular Matrix, as well as toactivation of Rho, leading to cell collapse by reorganization of thecytoskeleton. See Kruger et al., Nature Rev. Mol. Cell Biol. 6:789-800(2005); Pasterkamp, TRENDS in Cell Biology 15:61-64 (2005)).

In lymphoid tissues CD72 is utilized as a low affinity (300nM) CD100receptor (Kumanogoh et al., Immunity 3:621-631 (2000)). B cells and APCsexpress CD72, and anti-CD72 antibodies have many of the same effects assCD100, such as enhancement of CD40-induced B cell responses and B cellshedding of CD23. CD72 is thought to act as a negative regulator of Bcell responses by recruiting the tyrosine phosphatase SHP-1, which canassociate with many inhibitory receptors. Interaction of CD100 with CD72results in the dissociation of SHP-1, and the loss of this negativeactivation signal. CD100 has been shown to promote T cell stimulationand B cell aggregation and survival in vitro. The addition ofCD100-expressing cells or sCD100 enhances CD40-induced B cellproliferation and immunoglobulin production in vitro, and accelerates invivo antibody responses (Ishida et al., Inter. Immunol. 15:1027-1034(2003); Kumanogoh and H. Kukutani, Trends in Immunol. 22:670-676(2001)). sCD100 enhances the CD40 induced maturation of DCs, includingup-regulation of costimulatory molecules and increased secretion ofIL-12. In addition, sCD100 can inhibit immune cell migration, which canbe reversed by addition of blocking anti-CD100 mouse antibodies(Elhabazi et al., J. Immunol. 166:4341-4347 (2001); Delaire et al., J.Immunol. 166:4348-4354 (2001)).

Sema4D is expressed at high levels in lymphoid organs, including thespleen, thymus, and lymph nodes, and in non-lymphoid organs, such as thebrain, heart, and kidney. In lymphoid organs, Sema4D is abundantlyexpressed on resting T cells but only weakly expressed on resting Bcells and antigen-presenting cells (APCs), such as dendritic cells(DCs).

Cellular activation increases the surface expression of CD100 as well asthe generation of soluble CD100 (sCD100). The expression pattern ofCD100 suggests that it plays an important physiological as well aspathological role in the immune system. CD100 has been shown to promoteB cell activation, aggregation and survival; enhance CD40-inducedproliferation and antibody production; enhance antibody response to Tcell dependent antigens; increase T cell proliferation; enhancedendritic cell maturation and ability to stimulate T cells; and isdirectly implicated in demyelination and axonal degeneration (Shi etal., Immunity 13:633-642 (2000); Kumanogoh et al., J Immunol 169:1175-1181 (2002); and Watanabe et al., J Immunol 167:4321-4328 (2001)).

CD100 knock out (CD100−/−) mice have provided additional evidence thatCD100 plays an important role in both humoral and cellular immuneresponses. There are no known abnormalities of non-lymphoid tissues inCD100−/− mice. Dendritic cells (DCs) from the CD100−/− mice have poorallostimulatory ability and show defects in expression of costimulatorymolecules, which can be rescued by the addition of sCD100. Micedeficient in CD100 (CD100−/−) fail to develop experimental autoimmuneencephalomyelitis induced by myelin oligodendrocyte glycoproteinpeptide, because myelin oligodendrocyte glycoprotein-specific T cellsare not generated in the absence of CD100 (Kumanogoh et al., J Immunol169:1175-1181 (2002)). A significant amount of soluble CD100 is alsodetected in the sera of autoimmunity-prone MRL/lpr mice (model ofsystemic autoimmune diseases such as SLE), but not in normal mice.Further, the levels of sCD100 correlate with levels of auto-antibodiesand increase with age (Wang et al., Blood 97:3498-3504 (2001)). SolubleCD100 has also been shown to accumulate in the cerebral spinal fluid andsera of patients with demyelinating disease, and sCD100 inducesapoptosis of human pluripotent neural precursors (Dev cells), and bothinhibits process extension and induces apoptosis of rat oligodendrocytesin vitro (Giraudon et al., J Immunol 172(2):1246-1255 (2004)). Thisapoptosis was blocked by an anti-CD100 MAb.

III. Anti-CD100 Antibodies

Antibodies that bind CD100 have been described the art. See, forexample, US Publ. No. 2008/0219971 A1, International Patent ApplicationWO 93/14125 and Herold et al., Int. Immunol. 7(1): 1-8 (1995), each ofwhich is herein incorporated in its entirety by reference.

The antibodies of the invention comprise anti-CD100 antibodies orantigen-binding fragments, variants, or derivatives thereof that bind toCD100, e.g., MAb 2503, MAb 67, and MAb 76. In certain embodiments theanti-CD100 antibodies bind human, murine, or both human and murineCD100. In other embodiments, the anti-CD100 antibodies block CD100binding to its receptor, e.g., Plexin-B.

In one embodiment, the present invention provides an isolated bindingmolecule, e.g., an antibody or antigen binding fragment thereof, whichspecifically binds to the same CD100 epitope as monoclonal antibody2503, 67, or 76. In another embodiment, the present invention providesan isolated binding molecule, e.g., an antibody or antigen bindingfragment thereof, which specifically binds to CD100, and competitivelyinhibits monoclonal antibody 2503, 67, or 76 from specifically bindingto CD100, e.g., human, murine, or both human and murine CD100.

In certain embodiments, the binding molecule of the invention has anamino acid sequence that has at least about 80%, about 85%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, orabout 95% sequence identity to the amino acid sequence for the referenceanti-CD100 antibody molecule. In a further embodiment, the bindingmolecule shares at least about 96%, about 97%, about 98%, about 99%, or100% sequence identity to the reference antibody.

In another embodiment, the present invention provides an isolatedantibody or antigen-binding fragment thereof comprising, consistingessentially of, or consisting of an immunoglobulin heavy chain variabledomain (VH domain), where at least one of the CDRs of the VH domain hasan amino acid sequence that is at least about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, or identical toCDR1, CDR2 or CDR3 of SEQ ID NO: 9 or 10.

In another embodiment, the present invention provides an isolatedantibody or antigen-binding fragment thereof comprising, consistingessentially of, or consisting of an immunoglobulin heavy chain variabledomain (VH domain), where at least one of the CDRs of the VH domain hasan amino acid sequence that is at least about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, or identical toSEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

In another embodiment, the present invention provides an isolatedantibody or antigen-binding fragment thereof comprising, consistingessentially of, or consisting of an immunoglobulin heavy chain variabledomain (VH domain), where at least one of the CDRs of the VH domain hasan amino acid sequence identical, except for 1, 2, 3, 4, or 5conservative amino acid substitutions, to SEQ ID NO: 6, SEQ ID NO: 7, orSEQ ID NO: 8.

In another embodiment, the present invention provides an isolatedantibody or antigen-binding fragment thereof comprising, consistingessentially of, or consisting of a VH domain that has an amino acidsequence that is at least about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or 100% identical to SEQ ID NO: 9 or SEQ ID NO: 10,wherein an anti-CD100 antibody comprising the encoded VH domainspecifically or preferentially binds to CD100.

In another embodiment, the present invention provides an isolatedantibody or antigen-binding fragment thereof comprising, consistingessentially of, or consisting of an immunoglobulin light chain variabledomain (VL domain), where at least one of the CDRs of the VL domain hasan amino acid sequence that is at least about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, or identical toCDR1, CDR2 or CDR3 of SEQ ID NO: 17 or 18.

In another embodiment, the present invention provides an isolatedantibody or antigen-binding fragment thereof comprising, consistingessentially of, or consisting of an immunoglobulin light chain variabledomain (VL domain), where at least one of the CDRs of the VL domain hasan amino acid sequence that is at least about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, or identical toSEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

In another embodiment, the present invention provides an isolatedantibody or antigen-binding fragment thereof comprising, consistingessentially of, or consisting of an immunoglobulin light chain variabledomain (VL domain), where at least one of the CDRs of the VL domain hasan amino acid sequence identical, except for 1, 2, 3, 4, or 5conservative amino acid substitutions, to SEQ ID NO: 14, SEQ ID NO: 15,or SEQ ID NO: 16.

In a further embodiment, the present invention includes an isolatedantibody or antigen-binding fragment thereof comprising, consistingessentially of, or consisting of a VL domain that has an amino acidsequence that is at least about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or 100% identical to SEQ ID NO: 17 or SEQ ID NO: 18,wherein an anti-CD100 antibody comprising the encoded VL domainspecifically or preferentially binds to CD100.

Suitable biologically active variants of the anti-CD100 antibodies ofthe invention can be used in the methods of the present invention. Suchvariants will retain the desired binding properties of the parentanti-CD100 antibody. Methods for making antibody variants are generallyavailable in the art.

Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Walker and Gaastra, eds. (1983)Techniques in Molecular Biology (MacMillan Publishing Company, NewYork); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel etal., Methods Enzymol. 154:367-382 (1987); Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S.Pat. No. 4,873,192; and the references cited therein; hereinincorporated by reference. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the polypeptideof interest may be found in the model of Dayhoff et al. (1978) in Atlasof Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), pp. 345-352, herein incorporated by reference in itsentirety. The model of Dayhoff et al. uses the Point Accepted Mutation(PAM) amino acid similarity matrix (PAM 250 matrix) to determinesuitable conservative amino acid substitutions. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred. Examples of conservative aminoacid substitutions as taught by the PAM 250 matrix of the Dayhoff et al.model include, but are not limited to, Gly⇄Ala, Val⇄Ile⇄Leu, Asp⇄Glu,Lys⇄Arg, Asn⇄Gln, and Phe⇄Trp⇄Tyr.

In constructing variants of the anti-CD100 binding molecule, e.g., anantibody or antigen-binding fragment thereof, polypeptides of interest,modifications are made such that variants continue to possess thedesired properties, e.g., being capable of specifically binding to aCD100, e.g., human, murine, or both human and murine CD100, e.g.,expressed on the surface of or secreted by a cell and having CD100blocking activity, as described herein. Obviously, any mutations made inthe DNA encoding the variant polypeptide must not place the sequence outof reading frame and preferably will not create complementary regionsthat could produce secondary mRNA structure. See EP Patent ApplicationPublication No. 75,444.

Methods for measuring anti-CD100 binding molecule, e.g., an antibody orantigen-binding fragment thereof, binding specificity include, but arenot limited to, standard competitive binding assays, assays formonitoring immunoglobulin secretion by T cells or B cells, T cellproliferation assays, apoptosis assays, ELISA assays, and the like. See,for example, such assays disclosed in WO 93/14125; Shi et al., Immunity13:633-642 (2000); Kumanogoh et al., J Immunol 169:1175-1181 (2002);Watanabe et al., J Immunol 167:4321-4328 (2001); Wang et al., Blood97:3498-3504 (2001); and Giraudon et al., J Immunol 172(2):1246-1255(2004), all of which are herein incorporated by reference.

When discussed herein whether any particular polypeptide, including theconstant regions, CDRs, VH domains, or VL domains disclosed herein, isat least about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or even about 100% identical to anotherpolypeptide, the % identity can be determined using methods and computerprograms/software known in the art such as, but not limited to, theBESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, to find thebest segment of homology between two sequences. When using BESTFIT orany other sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

For purposes of the present invention, percent sequence identity may bedetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (1981) Adv. Appl. Math.2:482-489. A variant may, for example, differ from a referenceanti-CD100 antibody (e.g., MAb 2503, 67 or 76) by as few as 1 to 15amino acid residues, as few as 1 to 10 amino acid residues, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The precise chemical structure of a polypeptide capable of specificallybinding CD100 and retaining the desired CD100 blocking activity dependson a number of factors. As ionizable amino and carboxyl groups arepresent in the molecule, a particular polypeptide may be obtained as anacidic or basic salt, or in neutral form. All such preparations thatretain their biological activity when placed in suitable environmentalconditions are included in the definition of anti-CD100 antibodies asused herein. Further, the primary amino acid sequence of the polypeptidemay be augmented by derivatization using sugar moieties (glycosylation)or by other supplementary molecules such as lipids, phosphate, acetylgroups and the like. It may also be augmented by conjugation withsaccharides. Certain aspects of such augmentation are accomplishedthrough post-translational processing systems of the producing host;other such modifications may be introduced in vitro. In any event, suchmodifications are included in the definition of an anti-CD100 antibodyused herein so long as the desired properties of the anti-CD100 antibodyare not destroyed. It is expected that such modifications mayquantitatively or qualitatively affect the activity, either by enhancingor diminishing the activity of the polypeptide, in the various assays.Further, individual amino acid residues in the chain may be modified byoxidation, reduction, or other derivatization, and the polypeptide maybe cleaved to obtain fragments that retain activity. Such alterationsthat do not destroy the desired properties (e.g., binding specificityfor CD100, binding affinity, and CD100 blocking activity) do not removethe polypeptide sequence from the definition of anti-CD100 antibodies ofinterest as used herein.

The art provides substantial guidance regarding the preparation and useof polypeptide variants. In preparing the anti-CD100 binding molecule,e.g., an antibody or antigen-binding fragment thereof, variants, one ofskill in the art can readily determine which modifications to the nativeprotein's nucleotide or amino acid sequence will result in a variantthat is suitable for use as a therapeutically active component of apharmaceutical composition used in the methods of the present invention.

The constant region of an anti-CD100 antibody may be mutated to altereffector function in a number of ways. For example, see U.S. Pat. No.6,737,056B1 and U.S. Patent Application Publication No. 2004/0132101A1,which disclose Fc mutations that optimize antibody binding to Fcreceptors.

In certain anti-CD100 antibodies, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as tumorlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well known immunological techniqueswithout undue experimentation.

Anti-CD100 antibodies of the invention also include derivatives that aremodified, e.g., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom specifically binding to its cognate epitope. For example, but notby way of limitation, the antibody derivatives include antibodies thathave been modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind an anti-CD100 polypeptide).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind at least oneepitope of a CD100 polypeptide) can be determined using techniquesdescribed herein or by routinely modifying techniques known in the art.

In certain embodiments, the anti-CD100 antibodies of the inventioncomprise at least one optimized complementarity-determining region(CDR). By “optimized CDR” is intended that the CDR has been modified andoptimized sequences selected based on the sustained or improved bindingaffinity and/or anti-CD100 activity that is imparted to an anti-CD100antibody comprising the optimized CDR. “Anti-CD100 activity” or “CD100blocking activity” can include activity which modulates one or more ofthe following activities associated with CD100: B cell activation,aggregation and survival; CD40-induced proliferation and antibodyproduction; antibody response to T cell dependent antigens; T cell orother immune cell proliferation; dendritic cell maturation;demyelination and axonal degeneration; apoptosis of pluripotent neuralprecursors and/or oligodendrocytes; induction of endothelial cellmigration; inhibition of spontaneous monocyte migration; binding to cellsurface plexin B 1; or any other activity association with soluble CD100or CD100 that is expressed on the surface of CD100+ cells. Anti-CD100activity can also be attributed to a decrease in incidence or severityof diseases associated with CD100 expression, including, but not limitedto, certain types of lymphomas, autoimmune diseases, inflammatorydiseases including central nervous system (CNS) and peripheral nervoussystem (PNS) inflammatory diseases, transplant rejections, and invasiveangiogenesis. Examples of optimized antibodies based on murineanti-CD100 MAbs BD16 and BB18, were described in US Publ. No.2008/0219971 A1, International Patent Application WO 93/14125 and Heroldet al., Int. Immunol. 7(1): 1-8 (1995), each of which are hereinincorporated by reference in their entirety. The modifications mayinvolve replacement of amino acid residues within the CDR such that ananti-CD100 antibody retains specificity for the CD100 antigen and hasimproved binding affinity and/or improved anti-CD100 activity.

IV. Polynucleotides Encoding Anti-CD100 Antibodies

The present invention also provides for nucleic acid molecules encodinganti-CD100 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable domain (VHdomain), where at least one of the CDRs of the VH domain has an aminoacid sequence that is at least about 80%, about 85%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or identical to apolynucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO: 4, or SEQ ID NO: 5.

In other embodiments, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin VH domain, where at least one ofthe CDRs of the VH domain is selected from the group consisting of: (a)a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6; (b)a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7; and(c) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VH domain that has an amino acid sequence thatis at least about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or 100% identical to a reference VH domain polypeptide sequencecomprising SEQ ID NO: 9 or SEQ ID NO: 10, wherein an anti-CD100 antibodycomprising the encoded VH domain specifically or preferentially binds toCD100.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable domain (VLdomain), where at least one of the CDRs of the VL domain has an aminoacid sequence that is at least about 80%, about 85%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or identical to apolynucleotide sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO: 12, and SEQ ID NO: 13.

In other embodiments, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin VL domain, where at least one ofthe CDRs of the VL domain is selected from the group consisting of: (a)a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14;(b) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15; and (c) a CDR3 comprising the amino acid sequence set forth in SEQID NO: 16.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VL domain that has an amino acid sequence thatis at least about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or 100% identical to a reference VL domain polypeptide sequencecomprising SEQ ID NO: 17 or SEQ ID NO: 18, wherein an anti-CD100antibody comprising the encoded VL domain specifically or preferentiallybinds to CD100.

Any of the polynucleotides described above may further includeadditional nucleic acids, encoding, e.g., a signal peptide to directsecretion of the encoded polypeptide, antibody constant regions asdescribed herein, or other heterologous polypeptides as describedherein. Also, as described in more detail elsewhere herein, the presentinvention includes compositions comprising one or more of thepolynucleotides described above.

In one embodiment, the invention includes compositions comprising afirst polynucleotide and second polynucleotide wherein said firstpolynucleotide encodes a VH domain as described herein and wherein saidsecond polynucleotide encodes a VL domain as described herein.Specifically a composition which comprises, consists essentially of, orconsists of a VH domain-encoding polynucleotide, as set forth in SEQ IDNO: 19 or SEQ ID NO: 20, and a VL domain-encoding polynucleotide, forexample, a polynucleotide encoding the VL domain as set forth in SEQ IDNO: 21 or SEQ ID NO: 22.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotides thatencode fusion polypeptides, Fab fragments, and other derivatives, asdescribed herein, are also contemplated by the invention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeieret al., Bio Techniques 17:242 (1994)), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an anti-CD100 antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention, may be generated from nucleic acid from a suitable source. Ifa clone containing a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the antibody may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, preferably poly A+RNA, isolatedfrom, any tissue or cells expressing the antibody or other anti-CD100antibody, such as hybridoma cells selected to express an antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence to identify, e.g., a cDNAclone from a cDNA library that encodes the antibody or other anti-CD100antibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe anti-CD100 antibody, or antigen-binding fragment, variant, orderivative thereof is determined, its nucleotide sequence may bemanipulated using methods well known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel etal., eds. (1998) Current Protocols in Molecular Biology (John Wiley &Sons, NY), which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

A polynucleotide encoding an anti-CD100 binding molecule, e.g., anantibody, or antigen-binding fragment, variant, or derivative thereof,can be composed of any polyribonucleotide or polydeoxyribonucleotide,which may be unmodified RNA or DNA or modified RNA or DNA. For example,a polynucleotide encoding anti-CD100 antibody, or antigen-bindingfragment, variant, or derivative thereof can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, a polynucleotide encoding an anti-CD100 binding molecule,e.g., an antibody, or antigen-binding fragment, variant, or derivativethereof can be composed of triple-stranded regions comprising RNA or DNAor both RNA and DNA. A polynucleotide encoding an anti-CD100 bindingmolecule, e.g., antibody, or antigen-binding fragment, variant, orderivative thereof, may also contain one or more modified bases or DNAor RNA backbones modified for stability or for other reasons. “Modified”bases include, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

V. Fusion Proteins and Antibody Conjugates

As discussed in more detail elsewhere herein, anti-CD100 bindingmolecules, e.g., antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof, may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalent and non-covalentconjugations) to polypeptides or other compositions. For example,anti-CD100 antibodies may be recombinantly fused or conjugated tomolecules useful as labels in detection assays and effector moleculessuch as heterologous polypeptides, drugs, radionuclides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

Anti-CD100 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, may include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodybinding anti-CD100. For example, but not by way of limitation, theantibody derivatives include antibodies that have been modified, e.g.,by glycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, etc. Additionally, the derivative may contain one or morenon-classical amino acids.

Anti-CD100 binding molecules, e.g., antibodies of the invention, orantigen-binding fragments, variants, or derivatives thereof, can becomposed of amino acids joined to each other by peptide bonds ormodified peptide bonds, i.e., peptide isosteres, and may contain aminoacids other than the 20 gene-encoded amino acids. For example,anti-CD100 antibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniquesthat are well known in the art. Such modifications are well described inbasic texts and in more detailed monographs, as well as in a voluminousresearch literature. Modifications can occur anywhere in the anti-CD100binding molecule, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini, or on moieties such ascarbohydrates. It will be appreciated that the same type of modificationmay be present in the same or varying degrees at several sites in agiven anti-CD100 binding molecule. Also, a given anti-CD100 bindingmolecule may contain many types of modifications. Anti-CD100 bindingmolecules may be branched, for example, as a result of ubiquitination,and they may be cyclic, with or without branching. Cyclic, branched, andbranched cyclic anti-CD100 binding molecule may result fromposttranslational natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins—Structure and Molecular Properties, T. E. Creighton,W. H. Freeman and Company, NY; 2nd ed. (1993); Johnson, ed. (1983)Posttranslational Covalent Modification of Proteins (Academic Press,NY), pgs. 1-12; Seifter et al., Meth. Enzymol. 182:626-646 (1990);Rattan et al., Ann. NY Acad. Sci. 663:48-62 (1992)).

The present invention also provides for fusion proteins comprising ananti-CD100 antibody, or antigen-binding fragment, variant, or derivativethereof, and a heterologous polypeptide. The heterologous polypeptide towhich the antibody is fused may be useful for function or is useful totarget the anti-CD100 polypeptide expressing cells.

In one embodiment, a fusion protein of the invention comprises, consistsessentially of, or consists of, a polypeptide having the amino acidsequence of any one or more of the VH domains of an antibody of theinvention or the amino acid sequence of any one or more of the VLdomains of an antibody of the invention or fragments or variantsthereof, and a heterologous polypeptide sequence.

In another embodiment, a fusion protein for use in the diagnostic andtreatment methods disclosed herein comprises, consists essentially of,or consists of a polypeptide having the amino acid sequence of any one,two, three of the CDRs of the VH domain of an anti-CD100 antibody, orfragments, variants, or derivatives thereof, or the amino acid sequenceof any one, two, three of the CDRs of the VL domain an anti-CD100antibody, or fragments, variants, or derivatives thereof, and aheterologous polypeptide sequence. In one embodiment, a fusion proteincomprises a polypeptide having the amino acid sequence of at least oneVH domain of an anti-CD100 antibody of the invention and the amino acidsequence of at least one VL domain of an anti-CD100 antibody of theinvention or fragments, derivatives or variants thereof, and aheterologous polypeptide sequence. Preferably, the VH and VL domains ofthe fusion protein correspond to a single source antibody (or scFv orFab fragment) that specifically binds at least one epitope of CD100. Inyet another embodiment, a fusion protein for use in the diagnostic andtreatment methods disclosed herein comprises a polypeptide having theamino acid sequence of any one, two, three or more of the CDRs of the VHdomain of an anti-CD100 antibody and the amino acid sequence of any one,two, three or more of the CDRs of the VL domain of an anti-CD100antibody, or fragments or variants thereof, and a heterologouspolypeptide sequence. Preferably, two, three, four, five, six, or moreof the CDR(s) of the VH domain or VL domain correspond to single sourceantibody (or scFv or Fab fragment) of the invention. Nucleic acidmolecules encoding these fusion proteins are also encompassed by theinvention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989);Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670(1990));L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); and IgEreceptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No.1448 (1991)).

As discussed elsewhere herein, anti-CD100 binding molecules, e.g.,antibodies of the invention, or antigen-binding fragments, variants, orderivatives thereof, may be fused to heterologous polypeptides toincrease the in vivo half life of the polypeptides or for use inimmunoassays using methods known in the art. For example, in oneembodiment, PEG can be conjugated to the anti-CD100 antibodies of theinvention to increase their half-life in vivo. See Leong et al.,Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weiret al., Biochem. Soc. Transactions 30:512 (2002).

Moreover, anti-CD100 binding molecules, e.g., antibodies of theinvention, or antigen-binding fragments, variants, or derivativesthereof, can be fused to marker sequences, such as a peptide tofacilitate their purification or detection. In preferred embodiments,the marker amino acid sequence is a hexa-histidine peptide, such as thetag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,Chatsworth, Calif., 91311), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made may be selected empirically tooptimize the secretion or binding characteristics of the fusion protein.DNA encoding the fusion protein is then transfected into a host cell forexpression.

Anti-CD100 binding molecules, e.g., antibodies of the present invention,or antigen-binding fragments, variants, or derivatives thereof, may beused in non-conjugated form or may be conjugated to at least one of avariety of molecules, e.g., to improve the therapeutic properties of themolecule, to facilitate target detection, or for imaging or therapy ofthe patient. Anti-CD100 binding molecules, e.g., antibodies of theinvention, or antigen-binding fragments, variants, or derivativesthereof, can be labeled or conjugated either before or afterpurification, or when purification is performed.

In particular, anti-CD100 antibodies of the invention, orantigen-binding fragments, variants, or derivatives thereof, may beconjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes,viruses, lipids, biological response modifiers, pharmaceutical agents,or PEG.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared,e.g., by reacting a binding polypeptide with an activated ester ofbiotin such as the biotin N-hydroxysuccinimide ester. Similarly,conjugates with a fluorescent marker may be prepared in the presence ofa coupling agent, e.g., those listed herein, or by reaction with anisothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of theanti-CD100 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, are prepared in an analogous manner.

The present invention further encompasses anti-CD100 binding molecules,e.g., antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, conjugated to a diagnostic ortherapeutic agent. The anti-CD100 antibodies, including antigen-bindingfragments, variants, and derivatives thereof, can be used diagnosticallyto, for example, monitor the development or progression of a disease aspart of a clinical testing procedure to, e.g., determine the efficacy ofa given treatment and/or prevention regimen. For example, detection canbe facilitated by coupling the anti-CD100 antibody, or antigen-bindingfragment, variant, or derivative thereof, to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, radioactive materials, positron emitting metals using variouspositron emission tomographies, and nonradioactive paramagnetic metalions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which canbe conjugated to antibodies for use as diagnostics according to thepresent invention. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, (β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, or ⁹⁹Tc.

An anti-CD100 binding molecule, e.g., an antibody, or antigen-bindingfragment, variant, or derivative thereof, may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells.

An anti-CD100 binding molecule, e.g., an antibody, or antigen-bindingfragment, variant, or derivative thereof, also can be detectably labeledby coupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged anti-CD100 binding molecule is then determinedby detecting the presence of luminescence that arises during the courseof a chemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

One of the ways in which an anti-CD100 antibody, or antigen-bindingfragment, variant, or derivative thereof, can be detectably labeled isby linking the same to an enzyme and using the linked product in anenzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)” Microbiological Associates Quarterly Publication,Walkersville, Md.; Diagnostic Horizons 2:1-7 (1978); Voller et al., J.Clin. Pathol. 31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523(1981); Maggio, ed. (1980) Enzyme Immunoassay, CRC Press, Boca Raton,Fla.; Ishikawa et al., eds. (1981) Enzyme Immunoassay (Kgaku Shoin,Tokyo). The enzyme, which is bound to the anti-CD100 antibody will reactwith an appropriate substrate, preferably a chromogenic substrate, insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the anti-CD100binding molecule, e.g., antibody, or antigen-binding fragment, variant,or derivative thereof, it is possible to detect the binding moleculethrough the use of a radioimmunoassay (RIA) (see, for example, Weintraub(March, 1986) Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques (The Endocrine Society), which isincorporated by reference herein). The radioactive isotope can bedetected by means including, but not limited to, a gamma counter, ascintillation counter, or autoradiography.

An anti-CD100 binding molecule, e.g., antibody, or antigen-bindingfragment, variant, or derivative thereof, can also be detectably labeledusing fluorescence emitting metals such as 152Eu, or others of thelanthanide series. These metals can be attached to the binding moleculeusing such metal chelating groups as diethylenetriaminepentacetic acid(DTPA) or ethylenediaminetetraacetic acid (EDTA).

Techniques for conjugating various moieties to an antibody (e.g., ananti-CD100 antibody), or antigen-binding fragment, variant, orderivative thereof, are well known, see, e.g., Amon et al. (1985)“Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy,”in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (AlanR. Liss, Inc.), pp. 243-56; Hellstrom et al. (1987) “Antibodies for DrugDelivery,” in Controlled Drug Delivery, ed. Robinson et al. (2nd ed.;Marcel Dekker, Inc.), pp. 623-53); Thorpe (1985) “Antibody Carriers ofCytotoxic Agents in Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological and Clinical Applications, ed. Pinchera et al., pp.475-506; “Analysis, Results, and Future Prospective of the TherapeuticUse of Radiolabeled Antibody in Cancer Therapy,” in MonoclonalAntibodies for Cancer Detection and Therapy, ed. Baldwin et al.,Academic Press, pp. 303-16 (1985); and Thorpe et al. (1982) “ThePreparation and Cytotoxic Properties of Antibody-Toxin Conjugates,”Immunol. Rev. 62:119-58.

VI. Expression of Antibody Polypeptides

DNA sequences that encode the light and the heavy chains of the antibodymay be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

Following manipulation of the isolated genetic material to provideanti-CD100 antibodies, or antigen-binding fragments, variants, orderivatives thereof, of the invention, the polynucleotides encoding theanti-CD100 antibodies are typically inserted in an expression vector forintroduction into host cells that may be used to produce the desiredquantity of anti-CD100 antibody.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody that binds to atarget molecule described herein, e.g., CD100, requires construction ofan expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule or a heavyor light chain of an antibody, or portion thereof (preferably containingthe heavy or light chain variable domain), of the invention has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing an antibody encoding nucleotide sequence aredescribed herein. Methods that are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors may includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

For the purposes of this invention, numerous expression vector systemsmay be employed. For example, one class of vector utilizes DNA elementsthat are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells that have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) synthesized as discussedabove. Of course, any expression vector that is capable of elicitingexpression in eukaryotic cells may be used in the present invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF 1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those that express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation that can be carried out, forexample, by robotic systems.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the anti-CD100 antibody has been prepared, the expressionvector may be introduced into an appropriate host cell. Introduction ofthe plasmid into the host cell can be accomplished by various techniqueswell known to those of skill in the art. These include, but are notlimited to, transfection (including electrophoresis andelectroporation), protoplast fusion, calcium phosphate precipitation,cell fusion with enveloped DNA, microinjection, and infection withintact virus. See, Ridgway (1988) “Mammalian Expression Vectors” inVectors, ed. Rodriguez and Denhardt (Butterworths, Boston, Mass.),Chapter 24.2, pp. 470-472. Typically, plasmid introduction into the hostis via electroporation. The host cells harboring the expressionconstruct are grown under conditions appropriate to the production ofthe light chains and heavy chains, and assayed for heavy and/or lightchain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques, and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

As used herein, “host cells” refers to cells that harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells that may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines that are best suitedfor the desired gene product to be expressed therein. Exemplary hostcell lines include, but are not limited to, CHO (Chinese Hamster Ovary),DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (humancervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVIwith SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38,R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK(hamster kidney line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). Host cell lines are typically available fromcommercial services, the American Tissue Culture Collection or frompublished literature.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theantibody molecule may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (1993) Current Protocols inMolecular Biology (John Wiley & Sons, NY); Kriegler (1990) “GeneTransfer and Expression” in A Laboratory Manual (Stockton Press, NY);Dracopoli et al. (eds) (1994) Current Protocols in Human Genetics (JohnWiley & Sons, NY) Chapters 12 and 13; Colberre-Garapin et al. (1981) J.Mol. Biol. 150:1, which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel (1987) “TheUse of Vectors Based on Gene Amplification for the Expression of ClonedGenes in Mammalian Cells in DNA Cloning” (Academic Press, NY) Vol. 3.When a marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding anti-CD100 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can also be expressedin non-mammalian cells such as insect, bacteria or yeast or plant cells.Bacteria that readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies (WO 02/096948A2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye and Inouye,Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke and Schuster, J. Biol.Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be usedto express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141(1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. Thisplasmid already contains the TRP1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12(1977)). The presence of the trp1 lesion as a characteristic of theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a preferred method for increasing the affinity ofantibodies of the invention is disclosed in U.S. Patent ApplicationPublication No. 2002 0123057 A1.

VII. Treatment Methods Using Therapeutic Anti-CD100 Antibodies

Methods of the invention are directed to the use of anti-CD100 bindingmolecules, e.g., antibodies, including antigen-binding fragments,variants, and derivatives thereof, to treat patients having a diseaseassociated with soluble CD100 secreted from or expressed onCD100-expressing cells. By “CD100-expressing cell” is intended normaland malignant cells expressing CD100 antigen. Methods for detectingCD100 expression in cells are well known in the art and include, but arenot limited to, PCR techniques, immunohistochemistry, flow cytometry,Western blot, ELISA, and the like.

Though the following discussion refers to diagnostic methods andtreatment of various diseases and disorders with an anti-CD100 antibodyof the invention, the methods described herein are also applicable tothe antigen-binding fragments, variants, and derivatives of theseanti-CD100 antibodies that retain the desired properties of theanti-CD100 antibodies of the invention, e.g., capable of specificallybinding CD100, e.g., human, mouse, or human and mouse CD100, and havingCD100 neutralizing activity.

In one embodiment, treatment includes the application or administrationof an anti-CD100 binding molecule, e.g., an antibody or antigen bindingfragment thereof, of the current invention to a patient, or applicationor administration of the anti-CD100 binding molecule to an isolatedtissue or cell line from a patient, where the patient has a disease, asymptom of a disease, or a predisposition toward a disease. In anotherembodiment, treatment is also intended to include the application oradministration of a pharmaceutical composition comprising the anti-CD100binding molecule, e.g., an antibody or antigen binding fragment thereof,of the current invention to a patient, or application or administrationof a pharmaceutical composition comprising the anti-CD100 bindingmolecule to an isolated tissue or cell line from a patient, who has adisease, a symptom of a disease, or a predisposition toward a disease.

The anti-CD100 binding molecules, e.g., antibodies or binding fragmentsthereof, of the present invention are useful for the treatment ofvarious malignant and non-malignant tumors. By “anti-tumor activity” isintended a reduction in the rate of malignant CD100-expressing cellproliferation or accumulation, and hence a decline in growth rate of anexisting tumor or in a tumor that arises during therapy, and/ordestruction of existing neoplastic (tumor) cells or newly formedneoplastic cells, and hence a decrease in the overall size of a tumorduring therapy. For example, therapy with at least one anti-CD100antibody causes a physiological response, for example, a reduction inangiogenesis, which is beneficial with respect to treatment of diseasestates associated with CD100-expressing cells in a human.

In one embodiment, the invention relates to anti-CD100 bindingmolecules, e.g., antibodies or binding fragments thereof, according tothe present invention for use as a medicament, in particular for use inthe treatment or prophylaxis of cancer or for use in a precancerouscondition or lesion. In certain embodiments, an anti-CD100 bindingmolecule, e.g., an antibody or binding fragment thereof, of theinvention is used for the treatment of a CD100 over-expressing cancer.In certain embodiments, an anti-CD100 binding molecule, e.g., anantibody or binding fragment thereof, of the invention is used for thetreatment of a CD100 over-expressing head and neck or colon cancer.

Further, anti-CD100 binding molecules, e.g., antibodies or bindingfragments thereof, of the present invention can also be used to inhibitangiogenesis for the treatment of pathological conditions dependent uponthe formation of new blood vessels, including tumor development andmacular degeneration. Angiogenesis is a complex multistep morphogeneticevent during which endothelial cells, stimulated by major determinantsof vascular remodeling, dynamically modify their cell-to-cell andcell-to-matrix contacts and move directionally to be reorganized into amature vascular tree (Bussolino et al., Trends Biochem Sci. 22:251-256(1997); Risau, Nature 386:671-674 (1997); Jain, Nat. Med. 9:685-693(2003)). The formation of new blood vessels is a key step during embryodevelopment, but it also occurs in adults in physiologic and inpathologic conditions, such as retinopathy, rheumatoid arthritis,ischemia, and particularly tumor growth and metastasis (Carmeliet, Nat.Med. 9:653-660 (2003)). This pathological formation of new blood vesselsis herein referred to as “invasive angiogenesis.” Basile et al., PNAS103(24):9017-9022 (2006)) demonstrated that, when shed from HNSCC cells,CD100 stimulates endothelial cell migration, which was prevented byCD100-blocking antibodies and by CD100 knockdown. CD100 overexpressionwas also noted in prostate, colon, breast, and lung cancers, suggestingthat expression of CD100 is a frequently used strategy by which a widevariety of carcinomas may promote angiogenesis.

In accordance with the methods of the present invention, at least oneanti-CD100 binding molecule, e.g., an antibody or antigen bindingfragment thereof, as defined elsewhere herein is used to promote apositive therapeutic response with respect to a malignant human cell. By“positive therapeutic response” with respect to cancer treatment isintended an improvement in the disease in association with theanti-tumor activity of these binding molecules, e.g., antibodies orfragments thereof, and/or an improvement in the symptoms associated withthe disease. That is, an anti-proliferative effect, the prevention offurther tumor outgrowths, a reduction in tumor size, a decrease in tumorvasculature, a reduction in the number of cancer cells, and/or adecrease in one or more symptoms associated with the disease can beobserved. Thus, for example, an improvement in the disease may becharacterized as a complete response. By “complete response” is intendedan absence of clinically detectable disease with normalization of anypreviously abnormal radiographic studies, bone marrow, and cerebrospinalfluid (CSF). Such a response must persist for at least one monthfollowing treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number of tumorcells present in the subject) in the absence of new lesions andpersisting for at least one month. Such a response is applicable tomeasurable tumors only.

Tumor response can be assessed for changes in tumor morphology (i.e.,overall tumor burden, tumor cell count, and the like) using screeningtechniques such as bioluminescent imaging, for example, luciferaseimaging, bone scan imaging, and tumor biopsy sampling including bonemarrow aspiration (BMA). In addition to these positive therapeuticresponses, the subject undergoing therapy with the anti-CD100 bindingmolecule, e.g., an antibody or antigen-binding fragment thereof, mayexperience the beneficial effect of an improvement in the symptomsassociated with the disease. For example, the subject may experience adecrease in the so-called B symptoms, e.g., night sweats, fever, weightloss, and/or urticaria.

The anti-CD100 binding molecules, e.g., antibodies or antigen bindingfragments thereof, described herein may also find use in the treatmentof inflammatory diseases and deficiencies or disorders of the immunesystem that are associated with CD100 expressing cells. Inflammatorydiseases are characterized by inflammation and tissue destruction, or acombination thereof. By “anti-inflammatory activity” is intended areduction or prevention of inflammation. “Inflammatory disease” includesany inflammatory immune-mediated process where the initiating event ortarget of the immune response involves non-self antigen(s), including,for example, alloantigens, xenoantigens, viral antigens, bacterialantigens, unknown antigens, or allergens. In one embodiment, theinflammatory disease is an inflammatory disorder of the peripheral orcentral nervous system. In another embodiment, the inflammatory diseaseis an inflammatory disorder of the joints.

Further, for purposes of the present invention, the term “inflammatorydisease(s)” includes “autoimmune disease(s).” As used herein, the term“autoimmunity” is generally understood to encompass inflammatoryimmune-mediated processes involving “self” antigens. In autoimmunediseases, self antigen(s) trigger host immune responses. An autoimmunedisease can result from an inappropriate immune response directedagainst a self antigen (an autoantigen), which is a deviation from thenormal state of self-tolerance. In general, antibodies (particularly,but not exclusively, IgG antibodies), acting as cytotoxic molecules oras immune complexes, are the principal mediators of various autoimmunediseases, many of which can be debilitating or life-threatening.

In one embodiment, the anti-CD100 binding molecule, e.g., an antibody orantigen binding fragment, of the invention is used to treat multiplesclerosis (MS). MS, also known as disseminated sclerosis orencephalomyelitis disseminata, is an autoimmune condition in which theimmune system attacks the central nervous system, leading todemyelination. The name multiple sclerosis refers to the scars(scleroses, also referred to as plaques or lesions) that form in thenervous system. MS lesions commonly involve white matter areas close tothe ventricles of the cerebellum, brain stem, basal ganglia and spinalcord, and the optic nerve. MS results in destruction ofoligodendrocytes, the cells responsible for creating and maintaining themyelin sheath. MS results in a thinning or complete loss of myelin and,as the disease advances, transection of axons.

Neurological symptom can vary with MS, and the disease often progressesto physical and cognitive disability. MS takes several forms, with newsymptoms occurring either in discrete attacks (relapsing forms) orslowly accumulating over time (progressive forms). Between attacks,symptoms may go away completely, but permanent neurological damage oftenresults, especially as the disease advances.

Neutralization of CD100 using an anti-CD100 monoclonal antibody of theinvention, e.g., MAb 2503, MAb 67 or MAb 76, may be used to reduce theseverity of MS through several different mechanisms, e.g., anti-CD100monoclonal antibodies may block immune maturation and activation byCD100 to reduce the rate of relapse by reducing secondary immuneresponses to CNS antigens, and anti-CD100 monoclonal antibodies mayblock the effect of soluble CD100 in mediating apoptosis ofoligodendrocytes in the CNS may reduce disease severity by reducingdemyelination.

In one embodiment, the anti-CD100 binding molecule, e.g., an antibody orantigen binding fragment, of the invention is used to treat arthritis.Arthritis, is an inflammatory disease of the joints, which can be causedby an autoimmune condition in which the immune system attacks thejoints. In certain embodiments, the arthritis is selected from the groupconsisting of osteoarthritis, gouty arthritis, ankylosing spondylitis,psoriatic arthritis, reactive arthritis, rheumatoid arthritis, juvenileonset rheumatoid arthritis, infectious arthritis, inflammatoryarthritis, septic arthritis, degenerative arthritis, arthritis mutilans,and lyme arthritis. In one embodiment, the arthritis is rheumatoidarthritis (RA).

The present invention includes methods of treating or preventingarthritis by administering to a subject an anti-CD100 binding moleculeof the invention, e.g., MAb 2503, MAb 67 or MAb 76. Methods of thepresent invention may reduce the pain, swelling, or stiffness associatedwith arthritis, e.g., rheumatoid arthritis. The present invention isalso directed to methods for improving joint performance, function, andhealth. In some embodiments of the present invention, treatment resultsin a decrease in arthritis severity scores, a decrease in arthritisseverity/area under curve, a decrease in histopathology parametersassociated with arthritis (inflammation, pannus, cartilage damage, andbone damage), a decrease in serum arachidonic acid levels, or a decreasein anti-collagen antibodies. In certain embodiments, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms associated with arthritis; prevention of arthritis; delay inthe onset of arthritis; reduced incidence of arthritis in a population;diminishment of the extent of the condition associated with arthritis;stabilization (i.e., not worsening) of the state of the condition,disorder or disease associated with arthritis; delay in onset or slowingof the condition, disorder or disease progression associated witharthritis; amelioration of the condition, disorder or disease state,remission (whether partial or total) of the condition, disorder ordisease associated with arthritis, whether detectable or undetectable;or enhancement or improvement of the condition, disorder or diseaseassociated with arthritis.

The method of the present invention can be administered to individualswho have arthritis or individuals who are at risk for developingarthritis. Thus, in some embodiments the invention is directed to amethod of treating a subject having normal joints, borderline arthriticjoints, or very arthritic joints, the method comprising administering ananti-CD100 binding molecule of the invention, e.g., MAb 2503, MAb 67 orMAb 76, to a subject as described herein. In some embodiments, themethod of the present invention can be used to treat chronic arthritisfor the remainder of the life of the subject.

In accordance with the methods of the present invention, at least oneanti-CD100 binding molecule, e.g., an antibody or antigen bindingfragment thereof, as defined elsewhere herein is used to promote apositive therapeutic response with respect to treatment or prevention ofan autoimmune disease and/or inflammatory disease. By “positivetherapeutic response” with respect to an autoimmune disease and/orinflammatory disease is intended an improvement in the disease inassociation with the anti-inflammatory activity, anti-angiogenicactivity, anti-apoptotic activity, or the like, of these antibodies,and/or an improvement in the symptoms associated with the disease. Thatis, an anti-proliferative effect, the prevention of furtherproliferation of the CD100-expressing cell, a reduction in theinflammatory response including but not limited to reduced secretion ofinflammatory cytokines, adhesion molecules, proteases, immunoglobulins(in instances where the CD100 bearing cell is a B cell), combinationsthereof, and the like, increased production of anti-inflammatoryproteins, a reduction in the number of autoreactive cells, an increasein immune tolerance, inhibition of autoreactive cell survival, reductionin apoptosis, reduction in endothelial cell migration, increase inspontaneous monocyte migration, reduction in and/or a decrease in one ormore symptoms mediated by stimulation of sCD100 or CD100-expressingcells can be observed. Such positive therapeutic responses are notlimited to the route of administration and may comprise administrationto the donor, the donor tissue (such as for example organ perfusion),the host, any combination thereof, and the like.

Clinical response can be assessed using screening techniques such asmagnetic resonance imaging (MRI) scan, x-radiographic imaging, computedtomographic (CT) scan, flow cytometry or fluorescence-activated cellsorter (FACS) analysis, histology, gross pathology, and blood chemistry,including but not limited to changes detectable by ELISA, RIA,chromatography, and the like. In addition to these positive therapeuticresponses, the subject undergoing therapy with the anti-CD100 bindingmolecule, e.g., an antibody or antigen-binding fragment thereof, mayexperience the beneficial effect of an improvement in the symptomsassociated with the disease.

The anti-CD100 binding molecules, e.g., antibodies or antigen bindingfragments thereof, can be used in combination with at least one othercancer therapy, including, but not limited to, surgery or surgicalprocedures (e.g. splenectomy, hepatectomy, lymphadenectomy,leukophoresis, bone marrow transplantation, and the like); radiationtherapy; chemotherapy, optionally in combination with autologous bonemarrow transplant, or other cancer therapy; where the additional cancertherapy is administered prior to, during, or subsequent to theanti-CD100 binding molecule, e.g., antibody or antigen binding fragmentthereof, therapy. Thus, where the combined therapies compriseadministration of an anti-CD100 binding molecule, e.g., an antibody orantigen binding fragment thereof, of the invention in combination withadministration of another therapeutic agent, as with chemotherapy,radiation therapy, other anti-cancer antibody therapy, smallmolecule-based cancer therapy, or vaccine/immunotherapy-based cancertherapy, the methods of the invention encompass coadministration, usingseparate formulations or a single pharmaceutical formulation, or andconsecutive administration in either order.

The anti-CD100 binding molecules, e.g., antibodies or binding fragmentsthereof, of the invention can be used in combination with any knowntherapies for autoimmune and inflammatory diseases, including any agentor combination of agents that are known to be useful, or which have beenused or are currently in use, for treatment of autoimmune andinflammatory diseases. Thus, where the combined therapies compriseadministration of an anti-CD100 binding molecules in combination withadministration of another therapeutic agent, the methods of theinvention encompass coadministration, using separate formulations or asingle pharmaceutical formulation, and consecutive administration ineither order. In some embodiments of the invention, the anti-CD100antibodies described herein are administered in combination withimmunosuppressive drugs or anti-inflammatory drugs, wherein the antibodyand the therapeutic agent(s) may be administered sequentially, in eitherorder, or simultaneously (i.e., concurrently or within the same timeframe).

In some other embodiments, the anti-CD100 binding molecules, e.g.,antibodies or antigen binding fragments thereof, of the invention may beused alone or in combination with immunosuppressive drugs to treatand/or prevent rheumatoid arthritis. Thus, in some embodiments where theanti-CD100 antibodies of the invention are used to treat rheumatoidarthritis, the antibodies may used in combination with suitableimmunosuppressive drugs. As discussed above, treatment effectiveness maybe assessed using any means and includes, but is not limited to,effectiveness as measured by clinical responses defined by the AmericanCollege of Rheumatology criteria, the European League of Rheumatismcriteria, or any other criteria. See for example, Felson et al.,Arthritis. Rheum. 38:727-35 (1995) and van Gestel et al., ArthritisRheum. 39:34-40 (1996).

In yet other embodiments, the anti-CD100 antibodies of the invention maybe used alone or in combination with immunosuppressive drugs to treatand/or prevent multiple sclerosis. Thus in some embodiments where theanti-CD100 antibodies of the invention are used to treat multiplesclerosis, the antibodies may used in combination with suitableimmunosuppressive drugs.

A further embodiment of the invention is the use of anti-CD100 bindingmolecule, e.g., antibodies or antigen binding fragments thereof, fordiagnostic monitoring of protein levels in tissue as part of a clinicaltesting procedure, e.g., to determine the efficacy of a given treatmentregimen. For example, detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

VIII. Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering the anti-CD100 binding molecule,e.g., antibodies, or antigen-binding fragments, variants, or derivativesthereof, of the invention to a subject in need thereof are well known toor are readily determined by those skilled in the art. The route ofadministration of the anti-CD100 binding molecule, e.g, antibody, orantigen-binding fragment, variant, or derivative thereof, may be, forexample, oral, parenteral, by inhalation or topical. The term parenteralas used herein includes, e.g., intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, rectal, or vaginaladministration. While all these forms of administration are clearlycontemplated as being within the scope of the invention, an example of aform for administration would be a solution for injection, in particularfor intravenous or intraarterial injection or drip. Usually, a suitablepharmaceutical composition for injection may comprise a buffer (e.g.acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate),optionally a stabilizer agent (e.g. human albumin), etc. However, inother methods compatible with the teachings herein, anti-CD100 bindingmolecules, e.g., antibodies, or antigen-binding fragments, variants, orderivatives thereof, of the invention can be delivered directly to thesite of the adverse cellular population thereby increasing the exposureof the diseased tissue to the therapeutic agent.

As discussed herein, anti-CD100 binding molecules, e.g., antibodies, orantigen-binding fragments, variants, or derivatives thereof, of theinvention may be administered in a pharmaceutically effective amount forthe in vivo treatment of CD100-expressing cell-mediated diseases such ascertain types of cancers, autoimmune diseases, inflammatory diseasesincluding central nervous system (CNS) and peripheral nervous system(PNS) inflammatory diseases, and invasive angiogenesis. In this regard,it will be appreciated that the disclosed binding molecules of theinvention will be formulated so as to facilitate administration andpromote stability of the active agent. Preferably, pharmaceuticalcompositions in accordance with the present invention comprise apharmaceutically acceptable, non-toxic, sterile carrier such asphysiological saline, non-toxic buffers, preservatives and the like. Forthe purposes of the instant application, a pharmaceutically effectiveamount of an anti-CD100 binding molecule, e.g., an antibody, orantigen-binding fragment, variant, or derivative thereof, conjugated orunconjugated, shall be held to mean an amount sufficient to achieveeffective binding to a target and to achieve a benefit, e.g., toameliorate symptoms of a disease or disorder or to detect a substance ora cell.

The pharmaceutical compositions used in this invention comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

Preparations for parenteral administration includes sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include, e.g., water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as, for example,antimicrobials, antioxidants, chelating agents, and inert gases and thelike.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences (Mack Publishing Co.)16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an anti-CD100 antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit such as thosedescribed in U.S. patent application Ser. No. 09/259,337. Such articlesof manufacture will preferably have labels or package inserts indicatingthat the associated compositions are useful for treating a subjectsuffering from, or predisposed to a disease or disorder.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in this invention may be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also may be administered by nasal aerosol or inhalation.Such compositions may be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of an anti-CD100 binding molecule, e.g., antibody, orfragment, variant, or derivative thereof, that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Thecomposition may be administered as a single dose, multiple doses or overan established period of time in an infusion. Dosage regimens also maybe adjusted to provide the optimum desired response (e.g., a therapeuticor prophylactic response).

In keeping with the scope of the present disclosure, anti-CD100antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may be administered to a human or other animalin accordance with the aforementioned methods of treatment in an amountsufficient to produce a therapeutic effect. The anti-CD100 antibodies,or antigen-binding fragments, variants, or derivatives thereof of theinvention can be administered to such human or other animal in aconventional dosage form prepared by combining the antibody of theinvention with a conventional pharmaceutically acceptable carrier ordiluent according to known techniques. It will be recognized by one ofskill in the art that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. Those skilled in the art will furtherappreciate that a cocktail comprising one or more species of anti-CD100binding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof, of the invention may prove to beparticularly effective.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of anti-CD100 binding molecule, e.g., antibody orantigen binding fragment thereof, that when administered brings about apositive therapeutic response with respect to treatment of a patientwith a disease to be treated.

Therapeutically effective doses of the compositions of the presentinvention, for treatment of CD100-expressing cell-mediated diseases suchas certain types of cancers, e.g., head and neck, prostate, colon,breast, and lung cancers; autoimmune diseases, e.g., arthritis, multiplesclerosis, inflammatory diseases including central nervous system (CNS)and peripheral nervous system (PNS) inflammatory diseases; and invasiveangiogenesis, vary depending upon many different factors, includingmeans of administration, target site, physiological state of thepatient, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human, but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages may betitrated using routine methods known to those of skill in the art tooptimize safety and efficacy.

The amount of at least one anti-CD100 binding molecule, e.g., antibodyor binding fragment thereof, to be administered is readily determined byone of ordinary skill in the art without undue experimentation given thedisclosure of the present invention. Factors influencing the mode ofadministration and the respective amount of at least one anti-CD100binding molecule, e.g., antibody, antigen-binding fragment, variant orderivative thereof include, but are not limited to, the severity of thedisease, the history of the disease, and the age, height, weight,health, and physical condition of the individual undergoing therapy.Similarly, the amount of anti-CD100 binding molecule, e.g., antibody, orfragment, variant, or derivative thereof, to be administered will bedependent upon the mode of administration and whether the subject willundergo a single dose or multiple doses of this agent.

The present invention also provides for the use of an anti-CD100 bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, in the manufacture of a medicament for treating anautoimmune disease and/or inflammatory disease, including, e.g.,arthritis, multiple sclerosis, CNS and PNS inflammatory diseases, or acancer.

The invention also provides for the use of an anti-CD100 bindingmolecule, e.g., antibody of the invention, or antigen-binding fragment,variant, or derivative thereof, in the manufacture of a medicament fortreating a subject for treating an autoimmune disease and/orinflammatory disease, or for treating a cancer, wherein the medicamentis used in a subject that has been pretreated with at least one othertherapy. By “pretreated” or “pretreatment” is intended the subject hasreceived one or more other therapies (e.g., been treated with at leastone other cancer therapy) prior to receiving the medicament comprisingthe anti-CD100 binding molecule, e.g., antibody or antigen-bindingfragment, variant, or derivative thereof. “Pretreated” or “pretreatment”includes subjects that have been treated with at least one other therapywithin 2 years, within 18 months, within 1 year, within 6 months, within2 months, within 6 weeks, within 1 month, within 4 weeks, within 3weeks, within 2 weeks, within 1 week, within 6 days, within 5 days,within 4 days, within 3 days, within 2 days, or even within 1 day priorto initiation of treatment with the medicament comprising the anti-CD100binding molecule, for example, the monoclonal antibody 2503 disclosedherein, or antigen-binding fragment, variant, or derivative thereof. Itis not necessary that the subject was a responder to pretreatment withthe prior therapy or therapies. Thus, the subject that receives themedicament comprising the anti-CD100 binding molecule, e.g., an antibodyor antigen-binding fragment, variant, or derivative thereof could haveresponded, or could have failed to respond (e.g., the cancer wasrefractory), to pretreatment with the prior therapy, or to one or moreof the prior therapies where pretreatment comprised multiple therapies.Examples of other cancer therapies for which a subject can have receivedpretreatment prior to receiving the medicament comprising the anti-CD100binding molecule, e.g., antibody or antigen-binding fragment, variant,or derivative thereof include, but are not limited to, surgery;radiation therapy; chemotherapy, optionally in combination withautologous bone marrow transplant, where suitable chemotherapeuticagents include, but are not limited to, those listed herein above; otheranti-cancer monoclonal antibody therapy; small molecule-based cancertherapy, including, but not limited to, the small molecules listedherein above; vaccine/immunotherapy-based cancer therapies; steroidtherapy; other cancer therapy; or any combination thereof.

IX. Diagnostics

The invention further provides a diagnostic method useful duringdiagnosis of CD100-expressing cell-mediated diseases such as certaintypes of cancers, autoimmune diseases, inflammatory diseases including,e.g., arthritis, multiple sclerosis, central nervous system (CNS) andperipheral nervous system (PNS) inflammatory diseases, and invasiveangiogenesis, which involves measuring the expression level of CD100protein or transcript in tissue or other cells or body fluid from anindividual and comparing the measured expression level with a standardCD100 expression level in normal tissue or body fluid, whereby anincrease in the expression level compared to the standard is indicativeof a disorder.

The anti-CD100 antibodies of the invention and antigen-bindingfragments, variants, and derivatives thereof, can be used to assay CD100protein levels in a biological sample using classical immunohistologicalmethods known to those of skill in the art (e.g., see Jalkanen, et al.,J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingCD100 protein expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA), immunoprecipitation, or Western blotting.Suitable assays are described in more detail elsewhere herein.

By “assaying the expression level of CD100 polypeptide” is intendedqualitatively or quantitatively measuring or estimating the level ofCD100 polypeptide in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level) or relatively (e.g.,by comparing to the disease associated polypeptide level in a secondbiological sample). Preferably, CD100 polypeptide expression level inthe first biological sample is measured or estimated and compared to astandard CD100 polypeptide level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” CD100 polypeptide level is known, it can be used repeatedlyas a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source of cellspotentially expressing CD100. Methods for obtaining tissue biopsies andbody fluids from mammals are well known in the art.

X. Immunoassays

Anti-CD100 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays that can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as Western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds,(1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.,NY) Vol. 1, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds, (1994) Current Protocols in Molecular Biology (John Wiley &Sons, Inc., NY) Vol. 1 at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al., eds, (1994) Current Protocols in Molecular Biology (John Wiley &Sons, Inc., NY) Vol. 1 at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (JohnWiley & Sons, Inc., NY) Vol. 1 at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody.

Anti-CD100 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention, additionally, can be employedhistologically, as in immunofluorescence, immunoelectron microscopy ornon-immunological assays, for in situ detection of CD100 protein orconserved variants or peptide fragments thereof In situ detection may beaccomplished by removing a histological specimen from a patient, andapplying thereto a labeled anti-CD100 antibody, or antigen-bindingfragment, variant, or derivative thereof, preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of CD100 protein, or conserved variants or peptidefragments, but also its distribution in the examined tissue. Using thepresent invention, those of ordinary skill will readily perceive thatany of a wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays for CD100 gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofbinding to CD100 or conserved variants or peptide fragments thereof, anddetecting the bound antibody by any of a number of techniques well knownin the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled anti-CD100 antibody,or antigen-binding fragment, variant, or derivative thereof. The solidphase support may then be washed with the buffer a second time to removeunbound antibody. Optionally the antibody is subsequently labeled. Theamount of bound label on solid support may then be detected byconventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-CD100 antibody, orantigen-binding fragment, variant, or derivative thereof may bedetermined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

There are a variety of methods available for measuring the affinity ofan antibody-antigen interaction, but relatively few for determining rateconstants. Most of the methods rely on either labeling antibody orantigen, which inevitably complicates routine measurements andintroduces uncertainties in the measured quantities.

Surface plasmon resonance (SPR) as performed on BIACORE® offers a numberof advantages over conventional methods of measuring the affinity ofantibody-antigen interactions: (i) no requirement to label eitherantibody or antigen; (ii) antibodies do not need to be purified inadvance, cell culture supernatant can be used directly; (iii) real-timemeasurements, allowing rapid semi-quantitative comparison of differentmonoclonal antibody interactions, are enabled and are sufficient formany evaluation purposes; (iv) biospecific surface can be regenerated sothat a series of different monoclonal antibodies can easily be comparedunder identical conditions; (v) analytical procedures are fullyautomated, and extensive series of measurements can be performed withoutuser intervention. BIAapplications Handbook, version AB (reprinted1998), BIACORE® code No. BR-1001-86; BIAtechnology Handbook, version AB(reprinted 1998), BIACORE® code No. BR-1001-84. SPR based bindingstudies require that one member of a binding pair be immobilized on asensor surface. The binding partner immobilized is referred to as theligand. The binding partner in solution is referred to as the analyte.In some cases, the ligand is attached indirectly to the surface throughbinding to another immobilized molecule, which is referred as thecapturing molecule. SPR response reflects a change in mass concentrationat the detector surface as analytes bind or dissociate.

Based on SPR, real-time BIACORE® measurements monitor interactionsdirectly as they happen. The technique is well suited to determinationof kinetic parameters. Comparative affinity ranking is simple toperform, and both kinetic and affinity constants can be derived from thesensorgram data.

When analyte is injected in a discrete pulse across a ligand surface,the resulting sensorgram can be divided into three essential phases: (i)Association of analyte with ligand during sample injection; (ii)Equilibrium or steady state during sample injection, where the rate ofanalyte binding is balanced by dissociation from the complex; (iii)Dissociation of analyte from the surface during buffer flow.

The association and dissociation phases provide information on thekinetics of analyte-ligand interaction (k_(a) and k_(d), the rates ofcomplex formation and dissociation, k_(d)/k_(a)=K_(D)). The equilibriumphase provides information on the affinity of the analyte-ligandinteraction (K_(D)).

BIAevaluation software provides comprehensive facilities for curvefitting using both numerical integration and global fitting algorithms.With suitable analysis of the data, separate rate and affinity constantsfor interaction can be obtained from simple BIACORE® investigations. Therange of affinities measurable by this technique is very broad rangingfrom mM to pM.

Epitope specificity is an important characteristic of a monoclonalantibody. Epitope mapping with BIACORE®, in contrast to conventionaltechniques using radioimmunoassay, ELISA or other surface adsorptionmethods, does not require labeling or purified antibodies, and allowsmulti-site specificity tests using a sequence of several monoclonalantibodies. Additionally, large numbers of analyses can be processedautomatically.

Pair-wise binding experiments test the ability of two MAbs to bindsimultaneously to the same antigen. MAbs directed against separateepitopes will bind independently, whereas MAbs directed againstidentical or closely related epitopes will interfere with each other'sbinding. These binding experiments with BIACORE® are straightforward tocarry out.

For example, one can use a capture molecule to bind the first Mab,followed by addition of antigen and second MAb sequentially. Thesensorgrams will reveal: (1) how much of the antigen binds to first Mab,(2) to what extent the second MAb binds to the surface-attached antigen,(3) if the second MAb does not bind, whether reversing the order of thepair-wise test alters the results.

Peptide inhibition is another technique used for epitope mapping. Thismethod can complement pair-wise antibody binding studies, and can relatefunctional epitopes to structural features when the primary sequence ofthe antigen is known. Peptides or antigen fragments are tested forinhibition of binding of different MAbs to immobilized antigen. Peptideswhich interfere with binding of a given MAb are assumed to bestructurally related to the epitope defined by that MAb.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W. H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freeman & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlag); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hal12003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Selection and Characterization of Antibodies Specificfor CD100

Mouse anti-CD100 antibodies recognizing human, monkey and murine CD100and were generated using the methods described below.

The “Line 1” cell line was derived from a spontaneous lung tumor in aBALB/c mouse. The inventors previously showed that injecting BALB/c micewith live Line 1 cells transfected with a foreign cDNA was an effectiveway to induce immune responses (unpublished data). The Line 1 cell linewas transfected with an expression plasmid encoding full length humanCD100 cDNA (SEQ ID NO: 23). A stable line expressing human CD100 wasisolated from the transfected cell line (Linel.CD100). CD100 deficientmice (BALB/c background, see Kumanogoh et al., J. Immunol. 169:1175-1181(2002)) were primed by immunization with purified mouse CD100-His (theextracellular domain of mouse CD100 with a C-terminal 6X his tag forpurification) emulsified in complete Freund's adjuvant (CFA). One weekfollowing this immunization, the mice were injected intramuscularly(i.m.) with 200,000 live Linel.CD100 cells. Nineteen days after theLinel.CD100 injection the mice were sacrificed and spleens wereharvested and fused with P3X63Ag8.653 fusion partner (ATCC# CRL-1580)following standard procedures to generate hybridomas. Hybridoma cloneswere screened by ELISA for binding to human and mouse CD100. Inparticular, a hybridoma cell line, specific for MAb 67, was preparedusing hybridoma technology. (Kohler et al., Nature 256:495 (1975);Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas, Elsevier, N.Y., pp. 571-681 (1981)). A large panel ofmouse CD100 specific antibodies was generated. Most of these antibodiesalso bound with high affinity to human CD100. Two hybridomas, clones67-2 and 76-1, produced monoclonal antibodies (called MAb 67 and MAb 76,respectively) that exhibited high affinity for both mouse and humanCD100 (See Table 2).

TABLE 2 Affinity measurements for mouse anti-CD100 MAbs Affinity forMouse Affinity for Human MAb Isotype CD100 (nM)* CD100 (nM)* 67 IgG11.00 5.7 76 IgG2b 0.33 0.12 *Affinity was measured using BIACORE ®surface plasmon resonance technology

An antibody cross-blocking ELISA demonstrated that MAb 67 and MAb 76recognize distinct epitopes on CD100. In addition, MAbs 67 and 76recognize an epitope that is distinct from that recognized by theindependently derived murine anti-human CD100 antibody BD16 (describedin US 2008/0219971 A1). Thus, two independent mouse anti-CD100antibodies, MAb 76 (see SEQ ID NOs: 25-40) and MAb 67 (see SEQ ID NOs:3-8; 10; 11-16; 18; 20 and 22), were generated.

Example 2 MAbs 67 and 76 Block Activity of Mouse and Human CD100 InVitro

CD100 specific antibodies were screened for their ability to inhibitCD100 binding in a fluorescence blocking assay. An illustration of themethod used to test whether the CD100 specific antibodies blocked CD100from binding to Plexin B1 is shown in FIG. 1. Human 293 cells weretransfected with cDNA encoding a CD100 receptor: Plexin B 1. A stablecell line expressing Plexin B1 was selected (293/Plexin). CD100-Hisbound to Plexin B1 on the cell surface was detected by flow cytometryusing a biotin conjugated His tag specific monoclonal antibody andstreptavidin-APC. When a tested anti-CD100 MAb was able to block bindingof CD100 to Plexin B 1, then less CD100-His was detected on the cell,resulting in lower fluorescence.

Forty nanograms (ng) of human or mouse CD100-His (C-terminal His tag)were incubated alone or with various concentrations of anti-CD100 MAbovernight at 4° C. The next morning, either (1) CD100 only or (2) CD100pre-incubated with an anti-CD100 MAb (67 or 76) was combined with the293/Plexin cells and incubated for 30 minutes on ice. CD100 that boundto the cell through Plexin B1 was detected using biotinylated polyclonalrabbit anti-His MAb, followed by streptavidin-APC, and cells wereanalyzed by flow cytometry. The results indicated that neutralization ofCD100 resulted in lower fluorescence. In particular, pre-incubatingCD100 with MAb 67 or MAb 76 resulted in lower fluorescence compared toCD100 only (FIG. 2). Increasing the concentration of anti-CD100 MAb 67or 76 from 0.156 μg/ml to 0.625 μg/ml resulted in a further reduction influorescence. Similar blocking of human CD100 was also observed (datanot shown). Thus, these results showed that MAb 67 and MAb 76 were bothable to block human and mouse CD100 from binding to Plexin Bl.

CD100/Plexin B1 signaling has been shown to induce detachment from theextracellular matrix and cell collapse by inducing reorganization ofactin filaments (See Kruger et al., Nature Reviews Molecular CellBiology 6:789-800 (2005)). A heterologous assay to determine cellulardetachment following binding to an extracellular matrix was used todetermine whether anti-CD100 antibodies could block CD100 induceddetachment of 293/Plexin cells from Fibronectin coated plates.

For the cell detachment assay, 293/plexin cells (normally grown as asuspension cell line) were plated at a density of 40,000 cells/well ontoa fibronectin coated 96 well plate and allowed to attach overnight. Two(2) μg/ml of mouse CD100-His (C-terminal His tag) was incubated alone orwith various concentrations of an anti-CD100 MAb (67 or 76) for 6 hoursat 4° C. CD100 samples were then brought up to room temperature beforeaddition to the 293/plexin cells. Cells were treated with CD100 or CD100pre-incubated with antibody for 30 minutes at 37 ° C., washed twice withPBS, and stained with crystal violet for 15 minutes. Cells were thenwashed twice with PBS and dried. Images were taken on a scanner fordocumentation. The crystal violet was then solubilized for 15 minutes atRT with 100μ1 of 33% glacial acetic acid, and pipetted into a new plate.Absorbance was read at 570nm. CD100 causes a reduction in the number ofcells attached to the plate, and thus a reduced absorbance, whileneutralization of CD100 results in an increase in absorbance.

As shown in FIG. 3, both MAb 67 and MAb 76 were able to block mouseCD100 mediated cell detachment and increase absorbance compared toisotype control. Similarly, both MAbs were also able to block celldetachment mediated by human CD100 (data not shown).

MAbs 67 and 76 blocked binding of CD100 to cell surface Plexin B1 andinduced detachment of 293/Plexin cells from Fibronectin coated plates.The results of the in vitro functional assays described above showedthat MAb 67 and MAb 76 were able to block the function of mouse andhuman CD100 in vitro.

Example 3 Evaluation of Anti-CD100 Monoclonal Antibodies in MouseDisease Models

Anti-CD100 neutralizing monoclonal antibodies (76 and 67) were tested invivo in the SJL EAE animal model. Relapsing experimental autoimmuneencephalomyelitis (R-EAE) is a CD4+ T cell-mediated diseasecharacterized by inflammation and demyelination within the centralnervous system. In SJL mice, R-EAE can be induced by immunization with apeptide epitope of proteolipid protein (PLP₁₃₉₋₁₅₁) (HSLGKWLGHPDKF; SEQID NO: 24). This model is characterized by a moderate to severe acuteparalytic phase followed by remission and subsequent relapses. Diseaseseverity was scored using the scale shown in Table 3.

TABLE 3 Evaluation of the EAE clinical signs Score Signs Description 0Normal behavior No neurological signs. 1 Distal limp tail The distalpart of the tail is limp and droopy. 1.5 Complete limp tail The wholetail is loose and droopy. 2 Righting reflex Animal has difficultiesrolling onto its feet when laid on its back. 3 Ataxia Wobbly walk - whenthe mouse walks the hind legs are unsteady. 4 Early paralysis The mousehas difficulties standing on its hind legs but still has remnants ofmovement. 5 Full paralysis The mouse can't move its legs at all, itlooks thiner and emaciated. 6 Moribund/Death Death.

SJL mice were immunized with 100 μg PLP₁₃₉₋₁₅₁ in CFA. Pertussis toxinwas administered on Day 0 and again 48 hours later. Mice were treatedwith 30 mg/kg MAb 76, 67 or isotype control antibody twice per weekstarting on Day 0, or once per week starting at Day 7. Scoring of EAEclinical signs was initiated from the 10th day post-EAE induction.

As shown in FIG. 4A, treatment with MAb 76 or MAb 67 reduced theseverity of EAE in SJL mice compared to mouse IgG control. The percentreduction in Group Mean Score (GMS) between Day 21 and the end of thestudy for MAbs 76 and 67 at 1×/week and 2×/week each is shown in FIG.4B. As shown in FIG. 4B, MAb 76 (1×/week) had a percent inhibition ofGMS of 35-40%; MAb 67 (1×/week) had a percent inhibition of GMS of65-70%; MAb 76 (2×/week) had a percent inhibition of GMS of 40-50%; andMAb 67 (2×/week) had a percent inhibition of GMS of 45-50%. Theseresults show that both MAb 67 and MAb 76 attenuated relapsing remittingEAE in SJL mice.

The SJL EAE study was repeated using MAb 76 at 30 mg/kg dose and dosing1×/week. The results further demonstrated that MAb 76 was able to reducethe severity of EAE in SJL mice compared to mouse IgG (data not shown).The percent reduction in Group Mean Score (GMS) between Day 21 and theend of the study was between 50-60%. Furthermore, the SJL EAE study wasrepeated using MAb 76 or MAb 67 at 30 mg/kg dosing 1×/week. Treatmentwith MAb 67 or MAb 76 resulted in a percent reduction in GMS of between30-40% between Day 18 and the end of the study. The results in FIG. 5Aand FIG. 5B further demonstrate that MAb 76 and MAb 67 were able toreduce the severity of EAE in SJL mice.

The SJL EAE study was repeated using MAb 67 at 30 mg/kg dosing 1×/weekwhere treatment started at day 7 post-immunization. Treatment with MAb67 starting at day 7 resulted in a percent reduction in GMS of about 50%between Day 12 and the end of the study. The results are shown in FIG.6.

The results from these in vivo studies demonstrate that neutralizationof CD100 using MAb 76 or MAb 67 reduced the clinical signs of EAE indifferent murine EAE experiments. The results for MAb 76 and MAb 67 weresimilar to one another in these experiments.

These results demonstrated that MAb 76 and MAb 67 have the ability toblock CD100 activity in vitro, and, importantly, the ability to reducethe severity of EAE in different dosing experiments in the mouse EAEmodel.

Example 4 Preparation of Chimeric and Humanized Anti-CD100 MonoclonalAntibodies

The murine monoclonal antibody clone 67, described above, has been shownto have the ability to neutralize both human and mouse CD100 in vitroand to ameliorate EAE in murine models in vivo.

The variable heavy (VH) and variable light (VK) genes were cloned fromthe clone 67 hybridoma and their sequence was determined. The amino acidsequences of the MAb 67 VH and VK genes are shown below with the CDR1,CDR2 and CDR3 regions underlined.

MAb 67 VH: (SEQ ID NO: 10)QVQLQQSGPELVKPGASVKISCKASGYSFSDYYMHWVKQSPENSLEWIGQINPTTGGASYNQKFKGKATLTVDKSSSTAYMQLKSLTSEESAVYYC TRYYYGRHFDVWGQGTTVTVSSMAb 67 VK: (SEQ ID NO: 18)DIVMTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSN EDPYTFGGGTKLEIK

The MAb 67 VH gene was cloned into a mammalian expression vector thatcontained the human gamma 4 heavy chain constant region coding sequence,creating a full length chimeric heavy chain. The MAb 67 VK was clonedinto a mammalian expression vector that had the human Kappa constantregion coding sequence, creating a full length chimeric light chain. Inorder to make a chimeric antibody, the expression vectors containing thechimeric heavy chain and the chimeric light chain were co-transfectedinto CHO-S cells. The monoclonal antibody that was produced was secretedfrom the cells and harvested after a 3-6 day expression period. Theresulting MAb was purified using Protein A chromatography andcharacterized. The resulting chimeric MAb (MAb 2368) was demonstrated tobe specific for CD100 by flow cytometry and by ELISA, and was shown tobe able to compete with murine MAb 67 for binding to CD100.Collectively, these data demonstrate that the correct VH and VK genesencoding the 67 MAb were isolated.

The MAb 67 variable CDR regions were used to create a humanizedmonoclonal antibody. The humanized VH and VK genes were respectivelycloned into vectors containing the human gamma 4 and human kappaconstant domains. The pairing of the humanized VH and the humanized VKcreated the IgG4/kappa MAb 2503. The amino acid sequences of thehumanized MAb 67 VH (H2160) and VK (L553) are shown below with the CDR1,CDR2 and CDR3 regions underlined.

Sequence of H2160: (SEQ ID NO: 9)QVQLVQSGAEVKKPGSSVKVSCKASGYSFSDYYMHWVRQAPGQGLEWMGQINPTTGGASYNQKFKGKATITVDKSTSTAYMELSSLRSEDTAVYYC ARYYYGRHFDVWGQGTTVTVSSSequence of L553: (SEQ ID NO: 17)DIVMTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSN EDPYTFGQGTKLEIK

Polynucleotides encoding the VH (Human Immunoglobulin gene with humangamma; H2160) and VK (Human Immunoglobulin gene with human kappa; L553)regions of MAb 2503 antibody were cloned into pCMV-Script (Stratagene)vectors and were deposited with the American Type Culture Collection(“ATCC”) on May 7, 2009, and given ATCC Deposit Numbers PTA-10004 andPTA-10005, respectively. The ATCC is located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, USA. The ATCC deposits were madepursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure.

Example 5 Characterization of Humanized Anti-CD100 Monoclonal Antibody2503

Specificity of MAb 2503 was characterized by ELISA and Flow Cytometry.MAb 2503 was tested in an epitope competition assay with MAb 67 and inan affinity determination assay using BIACORE® surface plasmon resonancetechnology.

The epitope specificity of MAb 2503 and MAb 67 were determined bycompetition ELISA. For the ELISA protocol, an ELISA plate was coatedwith CD100-Fc. Mouse 67 or humanized 2503 MAbs were titrated. Theantibodies were allowed to bind to CD100 and unbound antibody was washaway. Biotinylated MAb 67 was added. The antibodies were allowed to bindto CD100 and unbound antibody was wash away. Biotinylated MAb 67 boundto CD100 was detected using Streptavidin-HRP. The ability of MAb 2503 orMAb 67 to block binding of biotinylated 67 to human CD100 (shown in FIG.7A) and mouse CD100 (shown in FIG. 7B) was analyzed by ELISA.

Binding affinities of anti-CD100 MAbs were determined using BIACORE®surface plasmon resonance technology. MAb 2503 was shown to only bindCD100 (human, mouse and marmoset) and CD100 expressing cell lines,indicating that MAb 2503 is specific for CD100. MAb 2503 showed anincreased affinity for human and mouse CD100 compared to MAb 67. Asummary of the affinity characteristics of MAb 2503 and MAb 67 ispresented below in Table 4.

TABLE 4 Affinities of anti-CD100 MAbs for human, marmoset and mouseCD100 Affinity for Human Affinity for Marmoset Affinity for Mouse MAbCD100 (nM)* CD100 (nM)* CD100 (nM)* 67 5.1 2.7 1.3 2503 5.4 2.4 1.5*Affinity was measured using BIACORE ® surface plasmon resonancetechnology

These assays show that MAb 2503 is CD100 specific and binds to the sameepitope as mouse MAb 67. MAb 2503 was shown to bind both mouse and humanCD100, thus an advantage of MAb 2503 is that it can be tested for safetyand efficacy in mice and also in humans. Furthermore, it was shown thatMAb 2305 bound to human, monkey, and mouse CD100.

Example 6 MAb 2503 Blocks Activity of Mouse and Human CD100 In Vitro

MAb 2503 was tested for the ability to block the function of CD100 usingthe assays described above in Example 2, including (1) the flowcytometry blocking assay and (2) the cell detachment assay.

CD100 specific antibodies, MAb 67 and MAb 2503, were screened for theirability to inhibit CD100 binding to Plexin B1 in a fluorescence blockingassay. The method used to test whether the CD100 specific antibodiesblocked CD100 from binding to Plexin B1 was shown in FIG. 1 (Example 2).Pre-incubating CD100 with MAb 67 or MAb 2503 resulted in lowerfluorescence compared to CD100 only. MAb 2503 was able to block bindingof human (see FIG. 8A), marmoset (see FIG. 8B) and mouse (see FIG. 8C)CD100 in vitro. Thus, these results showed that MAb 67 and MAb 2503 wereboth able to block human, monkey and mouse CD100 from binding to PlexinB1.

The ability of anti-CD100 MAbs to block human and monkey CD100 mediateddetachment of 293/Plexin cells from a fibronectin coated plate wasdetermined using the cell detachment assay described in Example 2. CD100causes a reduction in the number of cells attached to the plate, andthus a reduced absorbance. Neutralization of human CD100 and marmosetCD100 with MAb2503 or MAb 67 resulted in an increase in absorbance asshown in FIG. 9A and FIG. 9B, respectively. MAb 2503 and MAb 67 alsoneutralized mouse CD100 in this assay (Data not shown).

MAb 2503 blocked binding of human, mouse and monkey CD100 to cellsurface Plexin B1 and induced detachment of 293/Plexin cells fromFibronectin coated plates. Thus, these results show that MAb 2503 wasable to functionally neutralize human, mouse and monkey CD100.

Example 7 Anti-CD100 Antibody Inhibits Angiogenesis In Vivo

CD100 is a potent pro-angiogenic molecule and activation of Plexin B1through CD100 binding transactivates c-Met and promotes the invasiveability of tumor cells and promotes angiogenesis.

To demonstrate the in vivo effect of the absence of CD100 on tumorgrowth, CT26 tumor cells were injected into the leg muscle of wild-typeBalb/c or CD100−/− mice and the resulting tumor growth was measured. Asshown in FIG. 10, the tumor volume in CD100−/− mice was decreasedcompared to wild-type Balb/c mice in this study. These resultsdemonstrated that a mouse colon cancer cell line (CT26) has impairedgrowth in an environment lacking CD100.

Next, the ability of MAb 67 to reduce the growth of CT26 tumor cells inwild-type Balb/c mice was tested. CT26 tumor cells were injected intothe leg muscle of wild-type Balb/c (n=48) or CD100−/− (n=9) mice.Following injection, the wild-type mice were split into 2 groups of 24mice each. One group was treated starting on Day 1 by i.p. injectionwith 1 mg MAb 67, and the other group was treated by i.p. injection with1 mg mouse IgG. Treatments were repeated every 7 days. The mice wereanalyzed for tumor growth. As shown in FIG. 11, treatment with MAb 67reduced the growth of CT26 tumors in Balb/c mice compared to the mouseIgG control group.

Thus, these results show that an antibody having the structural andfunctional characteristics of MAb 67 reduced tumor growth in vivo.

Example 8 Anti-CD100 Antibody Inhibits Collagen Induced Arthritis InVivo

MAb 67 was tested for its ability to reduce arthritis is the CollagenInduced Arthritis (CIA) mouse model. The collagen-induced arthritis(CIA) model is a preclinical animal inflammation model of rheumatoidarthritis (RA) that is widely used to address disease pathogenesis andvalidate therapeutic RA targets (Brand et al., Nat. Protoc. 2(5):1269-75(2007)). The general CIA procedure is illustrated in FIG. 12 (anymodifications to this general procedure are described below). Briefly,arthritis was induced at day 0 by intradermal tail injection of anemulsion comprising collagen and Complete Freund's Adjuvant (CFA).Thereafter, the control and test treatments were administered bysubcutaneous (s.c.) or intraperitoneal (i.p.) injection twice weeklystarting on day 20. The test groups for Study 1 are described below inTable 5.

TABLE 5 Collagen Induced Arthritis (CIA) Study 1 test groups Group # An-# imals Treatment Dose Treatment Days 1 10 Mouse IgG 600 μg i.p. 2X/weekstarting on Day 20 Isotype control 2 10 MAb 67 600 μg i.p. 2X/weekstarting on Day 20 3 10 etanercept 600 ug s.c. 2X/week starting on Day20

The disease severity was scored using the scale shown in Table 6. Eachmouse paw received a score (macroscopic signs of arthritis wereevaluated 3-times weekly). The Arthritic Index (AI) was calculated byaddition of individual paw scores (maximum AI=16). Typically, the firstsigns of arthritis appear in the CIA model 21-28 days afterimmunization.

TABLE 6 CIA Scoring Method Score Description 0 no visible effects ofarthritis 1 edema and/or erythema of 1 digit 2 edema and/or erythema of2 digits 3 edema and/or erythema of more than 2 digit 4 severe arthritisof entire paw and digits

The results of Study 1 showed a reduction in arthritis diseasedevelopment in the CIA model in groups treated with 600 μg MAb 67.Arthritic Index (AI) in mice treated with 600 μg MAb 67 was compared toAI in mice treated with 600 μg negative control (IgG1) and 600 μgpositive control (etanercept) where treatment was started at day 20.Results are shown in FIG. 13A. These results show that MAb 67 was asgood as or better than etanercept at reducing arthritis diseasedevelopment in the CIA model.

A second study (Study 2) included MAb 67 and positive control treatmentswhere the treatment was delayed, i.e., started when the Arthritis Indexwas ≧3. The test groups for Study 2 are described below in Table 7.

TABLE 7 Collagen Induced Arthritis (CIA) Study 2 test groups Group # An-# imals Treatment Dose Treatment Days 1 10 Mouse IgG 600 μg i.p. 2X/weekstarting on Day 20 Isotype control 2 10 MAb 67 600 μg i.p. 2X/weekstarting on Day 20 3 10 MAb 67 600 μg i.p. 2X/week starting whenArthritis Index is ≧3 4 10 etanercept 600 ug s.c. 2X/week starting whenArthritis Index is ≧3

For Study 2, the AI results for treatment with MAb 67 (treatment startedat day 20 or when the AI was ≧3) were compared to treatment with anegative control (IgG1) and positive control (etanercept; treatmentstarted when the AI was ≧3). The results are shown in FIG. 13B. Theseresults show that MAb 67 was as good as or better than etanercept atreducing arthritis disease development in the CIA model when treatmentwas started at day 20 or delayed to AI≧3. Thus, MAb 67 was shown toprevent arthritis disease development, as well as treat arthritisdisease when administered once AI was ≧3 in the CIA model.

Example 9 Anti-CD100 Antibody Blocks Primary and Memory B Cell ResponsesIn Vivo

Balb/c and CD100−/− mice were immunized with (4-hydroxy-3-nitrophenyl)acetyl conjugated chicken gamma globulin precipitated with alum(aluminum-/magnesium-hydroxide) (“NP-CGG”) followed by treatment withcontrol IgG1 (Balb/c and CD100−/−) or MAb 67 (Balb/c only). The testgroups are shown below in Table 8.

TABLE 8 Primary and memory B-cell response test groups Group Strain #animals challenge Ab treatment dose Ab Injection 1 CD100 −/− 6 N/A N/AN/A N/A 2 CD100 −/− 6 NP-CGG/Alum Control IgG1 N/A Day (−7), 0, 7, 14,21, 28 3 Balb/c 6 N/A N/A N/A N/A 4 Balb/c 6 NP-CGG/Alum Control IgG1600 ug Day (−7), 0, 7, 14, 21, 28 5 Balb/c 6 NP-CGG/Alum MAb 67 600 ugDay (−7), 0, 7, 14, 21, 28

The mice were treated as described above in Table 8 starting on Day -7and were immunized with NP-CGG on Day 0. Three mice per group wereeuthanized on Day 10, and the spleen and lymph nodes were analyzed forgerminal center (GC) B cells (“B220+CD38lowPNA+”). Each spleen wasanalyzed separately, and the lymph nodes from all three mice werecombined into one sample for analysis. Treatment of the remaining micewas continued as shown in Table 8. On Day 21 the mice were boosted withthe same NP-CGG in Alum. On Day 31 the remaining mice were euthanizedand the spleen and lymph nodes were analyzed for germinal center (GC) Bcells (“B220+CD38lowPNA+”). Each spleen was analyzed separately, and thelymph nodes from all three mice were combined into one sample foranalysis. The results in FIG. 14A and FIG. 14B show that treatment with600 μg MAb 67 decreased the number of GC B cells in spleen (SP) andlymph nodes (LN) after both primary immunization (14A) and secondaryimmunization (14B), respectively. Thus, MAb 67 was shown to blockprimary and memory B cell responses in vivo.

Example 10 Anti-CD100 Antibody Slows Tumor Growth In Vivo

Reduction of tumor growth by MAb 67 was tested in CT26 and BCA34 mousexenograft tumor models. For these studies, tumor cells were injectedintramuscularly into the legs of Balb/c mice. Starting 1 day post-graft,mice were treated by intraperitoneal (i.p.) injection lx per week with 1mg MAb 67 antibody or negative control (IgG) in 0.2 ml volume. Change intumor volume (mm³) and/or thigh volume (mm³) was measured to determinetumor growth rates. The tumor growth rate for MAb 67 treated micecompared to the tumor growth rate in negative control mice was used tocalculate tumor growth delay (TGD).

The ability of BCA34 and EMT6 tumor cells to grow in an environmentlacking CD100 was also tested. For these studies, tumor cells wereinjected into the legs of Balb/c and CD100−/− (SEMA4D−/−) mice and tumorgrowth was measured.

CT26 Colon Tumor Cells

CT26 tumor cells are derived from murine colon tumors and express verylow levels of CD100. 50,000 CT26 colon tumor cells were injected s.c.into wild-type Balb/c mice (20 mice/treatment group). The change intumor volume (mm³) was measured in mice treated with 1 mg MAb 67 weeklystarting on Day 1 post-graft compared to mice injected with IgG control.The results (mean tumor volume over time) are shown in FIG. 15. The TGDfor mice treated with MAb 67 was 17% (p=0.0317). These results show thatCT26 tumor growth was reduced in MAb 67 treated mice.

BCA34 Fibroblastic Tumor Cells

BCA34 tumor cells express low levels of CD100. First, 50,000 BCA34 tumorcells were s.c. injected into the abdominal region of wild-type Balb/cmice or CD100−/− (“SEMA4D−/−”) mice. The change in tumor volume (mm³)was measured in these mice, and the results are shown in FIG. 16A. Theseresults showed that BCA34 tumor cells had impaired growth in anenvironment lacking CD100.

In a separate experiment, 50,000 BCA34 tumor cells were injected intothe leg muscles of wild-type Balb/c mice (21 mice/treatment group). Thechange in tumor volume (mm³) was measured in mice treated with 1 mg MAb67 weekly starting on Day 1 post-graft compared to mice injected withIgG control. The results (mean thigh volume over time) are shown in FIG.16B. The TGD for mice treated with MAb 67 was 18% (p=0.009). Theseresults show that BCA34 tumor growth was reduced in MAb 67-treated mice.

EMT6 Mammary Carcinoma Tumor Cells

EMT6 tumor cells are derived from murine mammary carcinoma tumors andexpress moderate levels of CD100. 50,000 EMT6 tumor cells were injectedinto the leg muscles of wild-type Balb/c mice or CD100−/− (“SEMA4D−/−”)mice. The change in tumor volume (mm³) was measured in these mice, andthe results are show in FIG. 17. Change in tumor volume (mm³) is shownfor wild-type Balb/c mice and CD100−/− mice (“SEMA4D−/−”). These resultsshowed that EMT6 tumor cells had impaired growth in an environmentlacking CD100.

Example 11 Anti-CD100 Antibody Slows Human Tumor Growth In Vivo

Reduction of tumor growth by MAb 2503 was tested in HN12 and HN6 HIF1amODD human xenograft tumor models. HN12 and HN6 HIF1a mODD are derivedfrom human head and neck tumors, and both xenografts express high levelsof HIF1a and CD100. The HN6 HIF-1a mODD xenograft model is described inSun et al., JBiol Chem 284(46):32066-74 (2009). For these experiments, 2tumors/mouse were s.c. injected into athymic nude mice (10mice/treatment group). Starting 1 day post-graft, mice were treated byi.p. injection 1× per week with 1 mg MAb 2503 antibody or negativecontrol (IgG4) in 0.2 ml volume. Change in tumor volume (mm³) wasmeasured. The tumor growth rate for MAb 2503-treated HN6 HIF-1a mODDmice compared to the growth rate in negative control mice was used tocalculate tumor growth delay (TGD).

The endpoint for this experiment was TGD. Tumor growth results for MAb2503 treatment in HN12 and HN6 HIF-1a mODD xenograft models are shown inFIG. 18 and FIG. 19A, respectively. The TGD in HN6 HIF-1a mODD xenograftmice treated with MAb 2503 was 13% (p=0.0008). Furthermore, pictures ofrepresentative tumors from HN6 HIF-1a mODD xenograft mice treated withIgG4 control and MAb 2505 are shown in FIG. 19B. These results show thattreatment with MAb 2503 reduced human head and neck tumor growth invivo.

Example 12 Stable CHO-S Cell Line for Expressing High Titers of MAb 2503

A stable CHO-S cell line was derived for expressing high titers of MAb2503. Complementary DNA (cDNA) for the heavy and light chains of thehumanized anti-CD100 monoclonal antibody 2503 were used to produceseveral Chinese hamster ovary (CHO-S) expression cell lines constructedusing GPEx™ technology (Catalent Pharma Solutions, Madison, Wis.) andprogenitor CHO-S cells (see Bleck et al., “An Alternative Method for theRapid Generation of Stable, High-Expressing Mammalian Cell Lines,”BioProcessing Journal (September/October 2005)).

Following single cell cloning, one expression clone was selected basedon growth in culture and antibody production, and a research bank ofvialed expression cells was produced. The expressed antibody produced bythis clone was characterized for potency and specificity using in vitroand in vivo functional assays (e.g., flow blocking and detachmentassays). Subsequently, cells from the selected CHO expression clone wereexpanded in culture and a second (Parental Seed Stock) bank prepared andfrozen. Cells from one vial from the parental seed stock bank weresubsequently expanded in culture and used for cGMP production of theMaster Cell Bank (MCB).

Example 13 Anti-CD100 Antibody Dosage and PK Studies in Rat andCynomolgus Monkey

Single intravenous injection saturation analysis of MAb 2503 in rat andcymonolgus monkey was performed.

Rat Study: 36 Sprague-Dawley rats (3/sex/group) were administered asingle intravenous injection of MAb 2503 ranging from 0.01 to 100 mg/kg.A flow cytometry-based saturation assay was performed on lysed wholeblood at various time points to determine the percent of the cellulartarget (SEMA4D) that was saturated with MAb 2503. There was good datacorrelation with the amount of MAb detected in the serum, suggestingthat saturation was dependent on the amount of free-drug in the serum,and that cells were saturated when approximately 1 to 5 ug/ml of freedrug is detected in the serum. The percent saturation of SEMA4D at MAb2503 doses of 0, 0.01, 0.1, 1.0, 10, and 100 mg/kg in male and femalerats is shown in FIG. 20A and FIG. 20B, respectively.

Primate Study: 28 cynomolgus monkeys (2/sex/group; 4/sex/control group)were administered a single intravenous injection of MAb 2503 rangingfrom 0.01 to 100 mg/kg. A flow cytometry-based saturation assay wasperformed on lysed whole blood at various time points to determine thepercent of the cellular target (SEMA4D) that was saturated with MAb2503. There was good data correlation with the amount of MAb detected inthe serum, suggesting that saturation was dependent on the amount offree-drug in the serum, and that cells were saturated when approximately1 to 5 ug/ml of free drug was detected in the serum. The percentsaturation of SEMA4D at MAb 2503 doses of 0, 0.01, 0.1, 1.0, 10, and 100mg/kg in male and female rats (combined) is shown in FIG. 21. The ratand primate saturation results were very similar.

Pharmacokinetic (PK) results including biological half-life (hours(days)), plasma antibody concentration at time zero following injection(C₀), and area under the plasma concentration curve from time zero totime t (AUC_(0-t)) for single intravenous injections of 0.01, 0.1, 1.0,10, or 100 mg/kg MAb 2503 in rat and primate are shown in Table 9.

TABLE 9 PK Values for 2503 antibody in rat and primate (cynomolgusmonkey) Dose Half-life (hours (days)) C₀ (μg/mL) AUC_(0-t) (mg/kg) CynoRat Cyno Rat Cyno Rat 0.01 0.1051 No data 0.3573 No data Phase A 0.1649(0.01) No data Phase B 43.21 (1.80) No data 0.1 3.356 1.99 23.09 11.40Phase A 0.1659 (0.01) 3.47 (0.14) Phase B 6.829 (0.28) No data 1.0 39.3534.14 1,974 834.7 Phase A 3.175 (0.13) 0.636 (0.026) Phase B 43.69(1.82) 27.77 (1.2) 10 327.1 317.7 38,741 27,431 Phase A 3.075 (0.13)5.85 (0.24) Phase B 109.4 (4.56) 105.7 (4.4) 100 3,681 3,305 477,154488,194 Phase A 6.978 (0.29) 9.20 (0.38) Phase B 239.8 (9.99) 246.2(10.3)

The MAb 2503 elimination phase half life was found to be similar betweenrat and cynomolgus monkey, and ranged from about 6 hours to about 10days in a dose dependent fashion. Both the saturation and PK resultsappear to be dose dependent and suggest a remarkably similar profilebetween rat and cynomolgus monkey. Furthermore, no significant toxicityfor MAb 2503 at doses ranging from 0.01 to 100 mg/kg was observed in rator cynomolgus monkey.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

What is claimed is:
 1. A method for treating an autoimmune disease or aninflammatory disease in an animal in need of treatment, comprisingadministering to an animal in need of treatment a compositioncomprising: (a) an isolated antibody or antigen binding fragment thereofthat specifically binds to CD100, wherein the antibody or fragmentthereof competitively inhibits a reference monoclonal antibodycomprising the heavy chain variable region (VH) amino acid sequence SEQID NO: 9 and the light chain variable region (VL) amino acid sequenceSEQ ID NO: 17 from specifically binding to CD100, or the antibody orfragment thereof specifically binds to the same epitope as a referencemonoclonal antibody comprising the heavy chain variable region (VH)amino acid sequence SEQ ID NO: 9 and the light chain variable region(VL) amino acid sequence SEQ ID NO: 17, and (b) a pharmaceuticallyacceptable carrier.
 2. The method of claim 1, wherein the antibody orfragment thereof comprises the Kabat heavy chain complementaritydetermining region-3 (VH-CDR3) amino acid sequence SEQ ID NO:
 8. 3. Themethod of claim 2, wherein the antibody or fragment thereof comprises aVH polypeptide comprising VH-CDR1, VH-CDR2, and VH-CDR3 amino acidsequences comprising SEQ ID NOs: 6, 7, and 8, respectively, and a VLpolypeptide comprising VL-CDR1, VL-CDR2, and VL-CDR3 amino acidsequences comprising SEQ ID NOs: 14, 15, and 16, respectively.
 4. Themethod of claim 2, wherein the VH of the antibody or antigen-bindingfragment thereof comprises an amino acid sequence at least 90% identicalto SEQ ID NO: 9 or SEQ ID NO:
 10. 5. The method of claim 2, wherein theVL of the antibody or antigen-binding fragment thereof comprises anamino acid sequence at least 90% identical to SEQ ID NO: 17 or SEQ IDNO:
 18. 6. The method of claim 2, wherein the VH and VL of the antibodyor antigen-binding fragment thereof comprise amino acid sequences atleast 90% identical to SEQ ID NO: 9 and SEQ ID NO: 17, respectively, orSEQ ID NO: 10 and SEQ ID NO: 18, respectively.
 7. The method of claim 6,wherein the VH and VL of the antibody or fragment thereof comprise theamino acid sequences SEQ ID NO: 9 and SEQ ID NO: 17, respectively, orSEQ ID NO: 10 and SEQ ID NO: 18, respectively.
 8. The method of claim 1,wherein the antibody or fragment thereof inhibits CD100 binding to aCD100 receptor.
 9. The method of claim 8, wherein the CD100 receptor isPlexin-B1.
 10. The method of claim 1, wherein the autoimmune disease orinflammatory disease is multiple sclerosis.
 11. The method of claim 1,wherein the autoimmune disease or inflammatory disease is arthritis. 12.The method of claim 11, wherein the autoimmune disease or inflammatorydisease is rheumatoid arthritis.
 13. The method of claim 11, wherein thetreatment results in a decrease in arthritis severity scores, a decreasein arthritis severity/area under curve, a decrease in histopathologyparameters associated with arthritis, a decrease in serum arachidonicacid levels, or a decrease in anti-collagen antibodies.
 14. The methodof claim 11, wherein the antibody or fragment thereof is administered incombination with an immunosuppressive drug.
 15. The method of claim 1,wherein the animal is a mammal.
 16. The method of claim 15, wherein themammal is a human.